Abstract In der Chromatographie gilt: Kleinere Partikel, bessere Trennleistung. Der steigende Rückdruck aber begrenzt den Teilchendurchmesser auf 3‐10 μm für die HPLC. Noch kleinere Partikel können bei der Ultra‐Performance‐LC verwendet werden ‐ der geringste Durchmesser beträgt derzeit 1,7 μm.
Monoclonal antibodies are typically glycosylated at asparagine residues in the Fc domain, and glycosylation heterogeneity at the Fc sites is well known. This paper presents a method for rapid analysis of glycosylation profile of the therapeutic monoclonal antibody trastuzumab from different production batches using electrospray quadrupole ion-mobility time-of-flight mass spectrometry (ESI-Q-IM-TOF). The global glycosylation profile for each production batch was obtained by a fast LC-MS analysis, and comparisons of the glycoprofiles of trastuzumab from different lots were made based on the deconvoluted intact mass spectra. Furthermore, the heterogeneity at each glycosylation site was characterized at the reduced antibody level and at the isolated glycopeptide level. The glycosylation site and glycan structures were confirmed by performing a time-aligned-parallel fragmentation approach using the unique dual-collision cell design of the instrument and the incorporated ion-mobility separation function. Four different production batches of trastuzumab were analyzed and compared in terms of global glycosylation profiles as well as the heterogeneity at each glycosylation site. The results show that each batch of trastuzumab shares the same types of glycoforms but relative abundance of each glycoforms is varied.
ADVERTISEMENT RETURN TO ISSUEPREVFeaturesNEXTAdvancing LC Performance with Smaller Particles and Higher PressureJeffrey R. Mazzeo, Uwe D.Neue, Marianna Kele, and Robert S. PlumbCite this: Anal. Chem. 2005, 77, 23, 460 A–467 APublication Date (Web):December 1, 2005Publication History Published online1 December 2005Published inissue 1 December 2005https://pubs.acs.org/doi/10.1021/ac053516fhttps://doi.org/10.1021/ac053516fnewsACS Publications. This publication is available under these Terms of Use. Request reuse permissions This publication is free to access through this site. Learn MoreArticle Views2265Altmetric-Citations276LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail PDF (353 KB) Get e-AlertscloseSupporting Info (4)»Supporting Information Supporting Information SUBJECTS:Chromatography Get e-Alerts
This chapter contains sections titled: Optimization of Enantiomer Separations in HPLC Introduction Basic Principles of Enantioselective HPLC Thermodynamic Fundamentals of Enantioselective HPLC Adsorption and Chiral Recognition Differences to Reversed-Phase and Normal-Phase HPLC Principles for Optimization of Enantioselective HPLC Separations Selectors and Stationary Phases Method Selection and Optimization Cellulose and Amylose Derivatives Immobilized Cellulose and Amylose Derivatives Stationary Phases Derived from Tartaric Acid π-Acidic and π-Basic Stationary Phases Macrocyclic Selectors, Cyclodextrins, and Antibiotics Proteins and Peptides Ruthenium Complexes Synthetic and Imprinted Polymers Metal Complexation and Ligand-Exchange Phases Chiral Ion Exchangers Avoiding Errors and Troubleshooting Equipment and Columns – Practical Tips Detection Mistakes Originating from the Analyte Preparative Enantioselective HPLC Determination of the Loading Capacity Determination of Elution Volumes and Flow Rates Enantiomer Separation using Simulated Moving Bed (SMB) Chromatography Principles of Simulated Moving Bed Chromatography Separation of Commercial Active Pharmaceutical Ingredients by SMB Enantioselective Chromatography by the Addition of Chiral Additives to the Mobile Phase in HPLC and Capillary Electrophoresis Determination of Enantiomeric Purity Through the Formation of Diastereomers Indirect Enantiomer Separation on a Preparative Scale Enantiomer Separations Under Supercritical Fluid Chromatographic (SFC) Conditions New Chiral Stationary Phases and Information Management Software Summary References Miniaturization mLC/NanoLC – Optimization and Troubleshooting Introduction Sensitivity Influence of Column Length Influence of Column Internal Diameter Influence of Stationary Phase Robustness System Choice Capillary Connections Precautions Against Blocking Testing for Leakages Guard Column Switching and Sample Loading Strategies Sensitivity/Resolution Column Dimensions Packing Materials/Surface Covering Detectors References Microchip-Based Liquid Chromatography – Techniques and Possibilities Introduction Techniques Pressure-Driven Liquid Chromatography (LC) Open-Channel Electrochromatography (OCEC) Packed-Bed Electrochromatography Microfabricated Chromatographic Beds (Pillar Arrays) In Situ Polymerized Monolithic Stationary Phases Optimization and Possibilities Separation Performance Isocratic and Gradient Elution Tailor-Made Stationary Phases Sample Pretreatment and More-Dimensional Separations Issues and Challenges Application Examples Conclusions and Outlook References Ultra-Performance Liquid Chromatography Introduction Isocratic Separations Gradient Separations References
Recently, sample preparation has been considered to be the major cause of bottlenecks during high-throughput analysis. With the assistance of robotic liquid handlers and the 96-well plate format, more samples can be prepared for subsequent liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis. Protein precipitation is still widely used despite potential loss of sensitivity or variable results due to ion suppression. The use of solid-phase extraction (SPE) clearly gives superior results but may not be as cost effective as protein precipitation due to the labor and material costs associated with the process. Here, a novel 96-well SPE plate is described that was designed to minimize the elution volume required for quantitative elution of analytes. The plate is packed with 2 mg of a high-capacity SPE sorbent that allows loading of up to 750 microL of plasma, while the novel design permits elution with as little as 25 microL. Therefore, the plate offers up to a 15-fold increase in sample concentration. The evaporation and reconstitution step that is typically required in SPE is avoided due to the concentrating ability of the plate. Examples of applications in drug discovery/development are shown and results are compared to protein precipitation. Excellent sensitivity and linearity are demonstrated.
'/%3e%3cuse xlink:href='%23a' transform='translate(0 -400)'/%3e%3cuse xlink:href='%23a' transform='translate(0 -600)'/%3e%3cuse xlink:href='%23a' transform='translate(0 -800)'/%3e%3cuse xlink:href='%23a' transform='translate(0 -1000)'/%3e%3cuse xlink:href='%23a' transform='translate(0 -1200)'/%3e%3cpath d='M0 0h1400v800H0z' style='fill:%23010066'/%3e%3cpath d='M576 100c-166.146 0-301 134.406-301 300s134.854 300 301 300c60.027 0 115.955-17.564 162.927-47.783-27.353 9.44-56.71 14.602-87.271 14.602-147.327 0-266.897-119.172-266.897-266.01 0-146.837 119.57-266.01 266.897-266.01 32.558 0 63.746 5.815 92.602 16.468C696.217 118.91 638.305 100 576 100z' style='fill:%23fc0'/%3e%3cpath d='m914.286 471.429-99.538-53.25 29.43 108.982-66.575-91.165-20.77 110.96-20.428-111.024-66.857 90.96L699.314 418l-99.701 52.943 74.065-85.192-112.8 4.441 103.694-44.62-103.555-44.94L673.8 305.42 600 220l99.538 53.25-29.43-108.982 66.575 91.165 20.77-110.96 20.428 111.023 66.857-90.959L814.97 273.43l99.702-52.944-74.065 85.193 112.8-4.441-103.694 44.62 103.555 44.94-112.785-4.79z' style='fill:%23fc0' transform='matrix(1.2738 0 0 1.2423 -89.443 -29.478)'/%3e%3c/svg%3e)
'/%3e%3c/defs%3e%3cpath fill='%23012169' d='M0 0h10080v5040H0z'/%3e%3cpath stroke='%23fff' stroke-width='.6' d='m0 0 6 3m0-3L0 3' clip-path='url(%23b)' transform='scale(840)'/%3e%3cpath stroke='%23e4002b' stroke-width='.4' d='m0 0 6 3m0-3L0 3' clip-path='url(%23c)' transform='scale(840)'/%3e%3cpath stroke='%23fff' stroke-width='840' d='M2520 0v2520M0 1260h5040'/%3e%3cpath stroke='%23e4002b' stroke-width='504' d='M2520 0v2520M0 1260h5040'/%3e%3cg fill='%23fff'%3e%3cuse xlink:href='%23d' x='2520' y='3780'/%3e%3cuse xlink:href='%23a' x='7560' y='4200'/%3e%3cuse xlink:href='%23a' x='6300' y='2205'/%3e%3cuse xlink:href='%23a' x='7560' y='840'/%3e%3cuse xlink:href='%23a' x='8680' y='1869'/%3e%3cuse xlink:href='%23e' x='8064' y='2730'/%3e%3c/g%3e%3c/svg%3e)
'%3e%3cpath fill='%23012169' d='M0 0v30h60V0z'/%3e%3cpath stroke='%23fff' stroke-width='6' d='m0 0 60 30m0-30L0 30'/%3e%3cpath stroke='%23C8102E' stroke-width='4' d='m0 0 60 30m0-30L0 30' clip-path='url(%23b)'/%3e%3cpath stroke='%23fff' stroke-width='10' d='M30 0v30M0 15h60'/%3e%3cpath stroke='%23C8102E' stroke-width='6' d='M30 0v30M0 15h60'/%3e%3c/g%3e%3c/svg%3e)