HALO BioClass Gives You the Right Column for Every Situation

Transcription

1 HALO BioClass Gives You the Right Column for Every Situation

2 HALO PROTEIN, PEPTIDE AND GLYCAN Today, researchers are keenly interested in both fast and high resolution separations of numerous biomolecules to support the development of novel therapeutic proteins and peptides in pharmaceutical drug development, to advance understanding in modern university laboratories, to characterize protein post-translation modifications, and to fully assess subtle differences in biosimilars and other products of bioengineering and manufacture. HALO BioClass columns have been developed to make such tasks easier and more effective, and are aimed at the following areas of bioseparations: I ntact proteins, monoclonal antibodies (mabs), biosimilars, and other large biomolecules such as pegylated proteins, antibody drug conjugates (ADCs), etc. Peptide mapping (analysis of enzyme digests) for characterization and monitoring of synthetic protein drugs Analysis of therapeutic peptides and peptide biomarkers (protein surrogates) H igh resolution separations of complex mixtures of glycans released from N- and O-linked glycoproteins Currently, the HALO BioClass column family is comprised of the following products. Their features and benefits are described below, and their specifications are shown in Table F. HALO Protein C Stability up to 9 C HALO Peptide ES-C Fast, high resolution separations C an elute very large proteins with good peak shape and recovery Extremely stable up to 9 C Compatible with UHPLC and HPLC Rugged, reliable performance Low LC-MS bleed Use with either UHPLC or HPLC HALO Protein ES-C Extremely stable up to 9 C HALO Peptide ES-CN Same benefits as Peptide ES-C C an elute very large proteins with good peak shape and recovery Alternative selectivity to Peptide ES-C for peptide mapping and proteomic applications Compatible with UHPLC and HPLC Very low LC-MS bleed High peak capacity HALO Glycan Improved retention of acidic and zwitterionic analytes Very low sensitivity to buffer concentration Able to separate isobaric oligosaccharides with different linkages

3 Table F. HALO BioClass Column Specifications Particle Size(s) (µm) Pore Size (Å) Carbon Load (%) Surface Area (m/g) Low ph Limit High ph Limit Max. Temp. Lower ph Limit Max. Temp. Upper ph Limit Endcapped HALO Protein C Yes HALO Protein ES-C.. 9 Yes HALO Peptide ES-C No HALO Peptide ES-CN Yes HALO Glycan No Structure of an antibody showing its heavy chain in blue and its light chain in green.

4 HALO Protein Ångstrom pores to provide unrestricted pore access for polypeptides and proteins as large as kda. µm Fused-Core particles with a very thin. µm outer porous shell - Provides narrower peaks and better recoveries for large biomolecules (vs. smaller pore sizes and non-core particles) Solid Core. µm - Allows HALO Protein columns to be used with both UHPLC and HPLC instrumentation for fast bioseparations at moderate back pressures C and sterically-protected ES-C phases. µm - Excellent high temperature stability (up to 9 C) for improved peak shape and recovery Shell with Å pores µm inlet frit Pressure limit, bar/9 psi LARGE PROTEIN SEPARATION USING HALO PROTEIN C FUSED-CORE COLUMN Figure DD. High resolution separation of light and heavy chains of a denatured contractile protein (whole myosin from purified rabbit skeletal muscle) using HALO Protein C at C. Light chain subunits ~- kda Absorbance (mau) Column:. x mm, HALO. µm Protein C Part Number: 9- Instrument: Agilent SL Injection Volume: µl Sample: denatured myosin Detection: nm Flow Rate:. ml/min. Mobile Phase A: water/.% TFA Mobile Phase B: acetonitrile/.9% TFA Gradient: % B in min. Temperature: C Heavy chain subunits ~ kda - Expanded view µm

5 HIGH RESOLUTION OF LIGHT AND HEAVY CHAIN VARIANTS OF IgG Masses deconvoluted using MagTran software* Column:. x mm,. μm HALO Protein C Part Number: 9- Mobile Phase A:.% formic acid with mm Ammonium Formate Mobile Phase B: % ACN/% IPA/% A solvent Gradient: 9 % B in min. Temperature: C Detection: nm and MS using pps scan rate from to m/z Injection Volume: μl of μg/μl reduced and alkylated lgg Sample Solvent:.% (v/v) formic acid in water MS Parameters: P ositive ion mode, ESI at +. kv, C heatblock, C capillary LC-MS System: Shimadzu Nexera and LCMS- (single quadrupole MS) m/z LC= light chain HC= heavy chain - LC:,9 LC:, HC:, HC:, HC:, HC:,9 LC:,. nm Figure EE. Very high resolution is obtained between variants of light and heavy chains of a reduced and alkylated monoclonal antibody (IgG) sample using a HALO Protein C column. 9. HC:,.... ULTRAFAST PROTEIN SEPARATION USING HALO PROTEIN ES-C. HALO Protein ES-C,. µm, Å Pressure: bar..9 Absorbance (mau) Figure FF. An ultrafast protein separation is achieved on a HALO Protein ES-C Ångstrom column in less than minute Non-core C, µm, Å Pressure: bar Column:. x mm HALO Protein ES-C Part Number: 9- Instrument: Agilent SL Injection Volume: μl Detection: nm Temperature: C Flow Rate: ml/min. Mobile Phase A: water/.% TFA Mobile Phase B: acetonitrile/.% TFA Gradient: % B in min. Using and µl heat exchangers in column compartment PEAK IDENTITIES. Ribonuclease A. Cytochrome c. Lysozyme. α-lactalbumin. Catalase NOTE: Peak widths at half height are shown above respective peaks. Comparative results presented here may not be representative for all applications. HALO PROTEIN C PROVIDES NARROWER AND TALLER PEAKS THAN TOTALLY POROUS COLUMN HALO Protein C,. µm, Å Pressure: bar Absorbance (mau) Figure GG. HALO Protein C dramatically outperforms a conventional non-core C column,delivering narrower and taller peaks with improved recovery of holotransferrin, apomyoglobin, catalase and enolase. - 9 Non-core C, µm, Å Pressure: bar PEAK IDENTITIES - Column:. x mm HALO Protein C Part Number: 9- Instrument: Agilent SL Injection Volume: μl Detection: nm Temperature: C Flow Rate:. ml/min. Mobile Phase A: water/.% TFA Mobile Phase B: acetonitrile/.% TFA Gradient: % B in min. 9. Ribonuclease A. Cytochrome c. Lysozyme. Holotransterrin. Apomyoglobin. Catalase. Enolase Peaks and were not detected. Comparative results presented here may not be representative for all applications. See page for full list of HALO Protein part numbers.

6 HALO Peptide HALO Angstrom columns currently available in three particle sizes (,. and μm) and in two sterically protected phases: ES-C (all particle sizes) and ES-CN (. and µm) (Table F) Extremely stable at high temperatures and low ph Ideal for both ultrafast and ultrahigh resolution separations of peptides and polypeptides up to kda Outperforms non-core μm, Angstrom columns in terms of peak width, peak capacity and peak height (Figure HH) Offers comparable peak capacity to sub- μm non-core columns at % back pressure (. µm) ~ % higher peak capacity than sub- µm non-core columns at comparable back pressure ( µm) µm Solid Core. µm. µm Shell with Å pores HALO. Peptide. µm Solid Core. µm. µm Shell with Å pores HALO Peptide C olumns (Peptide. and μm) can be used in series to increase peak capacity for UHPLC and HPLC analyses of complex tryptic digest samples (Figure II) H ALO Peptide ES-CN (. and µm) offers different selectivity and improved retention for polar peptides (Figure JJ). µm Solid Core. µm μm inlet frit (. and µm); µm inlet frit ( µm) provides extra protection from plugging P ressure limit, bar/9 psi (. and µm); Pressure limit, bar/ psi ( µm). µm Shell with Å pores Graphical representation of a polypeptide structure having beta-pleated sheet and alpha-helical regions

7 COMPARISON OF FUSED-CORE TO NON-CORE COLUMNS FOR PEPTIDE SEPARATIONS Figure HH. HALO Peptide. column produces significantly taller peaks and higher peak capacity than a non-core µm column.... Non-core C µm, Å Pmax = bar..... Columns:. x mm, HALO. Peptide ES-C and. x mm, non-core µm C column Part Number: 9- Mobile Phase A: 9% water/% ACN/.% TFA Mobile Phase B: % water/% ACN/.% TFA Gradient: -.% B in min. Flow Rate:. ml/min. Temperature: C Injection Volume: μl LC System: Agilent. Absorbance (mau) PEAK IDENTITIES: Gly-Tyr. Angiotensin / (-) amide. Val-Tyr-Val. Met-Enk. Angiotensin / (-) amide. Angiotensin II. Leu-Enk. Angiotensin (-) human 9. Angiotensin (-) mouse. HALO Peptide ES-C. µm, Å Pmax = bar NOTE: Peak widths at half height are shown above respective peaks. Comparative results presented here may not be representative for all applications. COUPLED HALO PEPTIDE COLUMNS FOR MAXIMUM PEAK CAPACITY Figure II. Three HALO Peptide ES-C -mm columns ( mm total length) were connected in series to achieve a peak capacity of for this mixture of tryptic digests of α--glycoprotein and apotransferrin... Absorbance (mau).. Columns: Three -mm. mm HALO Peptide. ES-C columns Part Number: of 9- Mobile Phase A: water/.% formic acid/ mm ammonium formate Mobile Phase B: A with % acetonitrile Gradient: % B in min. Flow Rate:. ml/min. Temperature: C Detection: nm Injection Volume: μl [ μg each] of α--glycoprotein tryptic digest and apotransferrin tryptic digest ALTERNATE SELECTIVITY USING HALO PEPTIDE ES-CN Figure JJ. HALO Peptide ES-CN offers alternative selectivity to HALO ES-C for this mixture of peptides and polypeptides. 9 Absorbance (mau) HALO Peptide ES-CN 9-9 HALO Peptide ES-C 9 PEAK IDENTITIES: Column:. x mm Part Numbers: HALO Peptide ES-CN: 9- HALO Peptide ES-C: 9- Instrument: Optimized Agilent Injection Volume: μl Detection: nm Temperature: C Flow Rate:. ml/min Mobile Phase A: water/.% TFA Mobile Phase B: ACN/.% TFA Gradient: % B in min. See page for full list of HALO Peptide part numbers.. Asp-Phe. Angiotensin (-) amide. Tyr-Tyr-Tyr. Bradykinin. Leu-Enk. Angiotensin II. Neurotensin. ß endorphin 9. Sauvagine. Mellitin

8 HALO Glycan 9 Ångstrom pore size Incorporates a highly polar ligand that contains hydroxyl groups tethered to. µm Fused-Core silica particles via novel, proprietary linkage chemistry (Table F). µm. µm Solid Core Ideal for hydrophilic interaction liquid chromatography (HILIC) separations of oligosaccharides, and particularly, of released and labeled glycans from glycoproteins and proteoglycans. µm Mobile phases typically consist of acetonitrile and aqueous ammonium formate buffer ( mm, ph.) used to form a gradient of increasing water content during elution Shell with 9 Å pores Each lot of HALO Glycan material is tested for quality assurance (Figure KK) by separation of a procainamidereducing-end-labeled glycan ladder of oligosaccharides having glucose units (GU). - Peaks for oligosaccharides composed of and GU must meet tight specifications for retention and peak width before lot is approved for glycan analysis µm inlet frit Pressure limit, bar/9 psi QA ANALYSIS OF HALO GLYCAN Figure KK. Example QA Chromatogram for HALO Glycan column. Each HALO Glycan packing lot is tested using this glycan ladder mixture to assess and ensure lot-to-lot reproducibility. G# = DP of maltooligosaccharide For example, G = maltotriose Column:. x mm,. μm HALO Glycan Part Number: 99- Mobile Phase A: mm Ammonium Formate, ph. Mobile Phase B: Acetonitrile Gradient: -% B in min. Flow Rate:. ml/min. Pressure: 9 bar Temperature: C Detection: UV nm Injection Volume: μl Sample Solvent: / ACN/water Response Time:. sec. Data Rate:. Hz Flow Cell:. μl semi-micro LC System: Shimadzu Nexera G G nm G G G G G9 G G G

9 SEPARATION OF N-LINKED GLYCANS FROM RIBONUCLEASE B Figure LL. Gradient HILIC-MS separation of N-linked glycans, which had been released using PNGase from ribonuclease B, using the HALO Glycan column. Man Ribonuclease B N-Linked Glycans Man Man Column:. x mm,. µm HALO Glycan Part Number: 99- Mobile Phase A: mm Ammonium Formate, ph. Mobile Phase B: Acetonitrile Gradient:..% B in. min Flow Rate:. ml/min. Temperature: C Detection: UV nm Injection Volume: μl Man9 Man SEPARATION OF N-LINKED GLYCANS FROM HUMAN IgG UV GF... GF GF Figure MM. Released- and procainamide-labeled glycans from human IgG were separated using a. x mm HALO Glycan column and detected using UV and selected-ion-monitoring MS detection. -., :.(+) :9.(+) :.(+) :.(+) :9.(+) :.(+) :.(+) :.(+) :.(+) :9.(+) :.(+) :9.(+) :9.(+) :.(+) :.(+) :.9(+) :9.(+),,,,, GF GF MS GF GFB Column:. x mm, HALO. µm Glycan Part Number: 99- Mobile Phase A: mm Ammonium Formate, ph. Mobile Phase B: Acetonitrile Gradient:. % B in min Flow Rate:. ml/min. Temperature: C Detection: UV nm Injection Volume: μl LC System: Shimadzu Nexera MS: Shimadzu LCMS (single quadrupole) ESI: +. kv Scan range: - m/z Scan rate: pps GF+NeuAc GF-N G G GFB G+NeuAc GF+NeuAc GFB+NeuAc GFB+NeuAc See page for full list of HALO Glycan part numbers. 9

10 HALO UHPLC AND HPLC GUARD COLUMNS Collect strongly retained compounds from the sample and minimizes column fouling F inger-tight, direct-connect units that auto-adjust to any column with a inlet port Ultra-low dispersion, easy to use, operate at pressures up to bar Easily replace guard cartridge without removing guard holder from the flow path A vailable for all HALO analytical geometries (.,. and. mm ID) and phases See figure below for an exploded view of the HALO guard cartridge and guard holder. Please see pages for ordering information. Finger-tight guard cartridge holder, Inlet Provides easy replacement of guard cartridge Finger-tight guard cartridge holder, Outlet Allows for replacement of guard cartridge without removing the guard holder from the flow path HALO guard cartridge All HALO phases available Titanium hybrid ferrule Adds durability for multiple connections Auto-adjusting zero dead volume (ZDV) end fitting Ensures optimum, ZDV connection to the analytical column The Optimize Technologies EXP Titanium Hybrid Ferrule is Patent Pending. The EXP Holder is patented under Patent No.,9,9 B. The OPTI- prefix is a registered trademark of Optimize Technologies, Inc.

11 HALO GUARD COLUMNS: PROTECTION + PERFORMANCE with guard column nm Figure NN. HALO guard columns provide optimum protection for your HALO HPLC and UHPLC column without sacrificing column efficiency no guard column Column:. x mm. µm HALO C Mobile Phase: / ACN/water Flow Rate:. ml/min. Temperature: C Detection: nm Injection Volume: µl Pressure: bar with guard column bar without guard column Instrument: Optimized Agilent bypassed semi-micro flow cell. ID tubing Hz data rate

12 HALO PROTEIN COLUMNS HALO GLYCAN COLUMNS Part numbers for nano, capillary, analytical and semipreparative HALO Protein columns in both C and ES-C phases are provided below. Guard columns are available for HALO Protein columns in.,. and. mm IDs for UHPLC and HPLC applications to provide additional column protection when desired. HALO Glycan columns are available in. and. mm diameters in the following lengths in. µm particle sizes. Guard columns are available for UHPLC and HPLC applications if additional protection is desired. Dimensions (in mm). x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x HALO Protein C HALO Protein ES-C HALO Protein C HALO Protein ES-C Guard Columns, -pack Dimensions (in mm). x. x. x Guard Column Holder 99- Dimensions (in mm). x. x. x. x. x. x HALO Glycan Guard Columns, -pack Dimensions (in mm). x. x Guard Column Holder HALO Glycan Visit our website

13 HALO PEPTIDE COLUMNS The part numbers are provided below for the nano, capillary, analytical and semi-preparative HALO. and HALO Peptide columns in both ES-C and ES-CN phases. Guard columns are available for.,. and. mm internal diameters for UHPLC and HPLC applications, if additional protection is desired. Dimensions ID x Length (in mm). x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x. x HALO Peptide ES-C HALO. Peptide ES-C HALO. Peptide ES-CN HALO- Peptide ES-C HALO- Peptide ES-CN HALO. Peptide ES-C HALO. Peptide ES-CN HALO- Peptide ES-C HALO- Peptide ES-CN Guard Columns, -pack Dimensions ID x Length (in mm). x. x. x Guard Column Holder HALO Peptide ES-C Visit our website

14 Halo and Fused-Core are registered trademarks of Advanced Materials Technology, Inc.