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The CHROMacademy Essential Guide Webcast:
Choosing the Correct Column and Mobile Phase for LC-MS Peptide Mapping

Thursday 21st September 2017,
11:00am EDT/ 8:00am PDT/ 4:00pm BST/ 5:00pm CEST

 


Agilent

This webcast will focus on essential parameters for successful peptide mapping by LC/MS. Obtaining complete sequence coverage as well as separation and detection of low-level post-translational modifications is a challenging endeavor. Guidance will be given on matching column, mobile phase, and detector for optimum sequence coverage. Additionally, optimization of key method conditions such as gradient length and temperature will be discussed.

Presented by

Presented by Dr. Anne Blackwell (Product Support Scientist, Agilent Technologies).

Topics Covered

  • Choosing the correct column for LC/MS peptide mapping
  • Impact of column chemistry on selectivity and resolution
  • Impact of mobile phases additives on sensitivity, selectivity, and peak capacity

Presenter Information »

 
 

Choosing the Correct Column and Mobile Phase for LC-MS Peptide Mapping - Tutorial

This month’s webcast focused on developing LC-MS peptide mapping methods - in particular column and mobile phase selection.  The CHROMacademy material provided in this month’s tutorial will give you further insight into how peptides are analyzed using a variety of chromatographic techniques (RP, HILIC) and MS detection.

Our Quick guides, CHROMacademy course content, and archived webcasts and tutorials will deliver further knowledge on the analysis of biologics at the protein and peptide level.

Watch the webcast

eLearning Modules


 
Introduction to Mass Spectrometry for Biomolecules

An introduction to the MS strategies used for biopharmaceutical characterization.

Introduction | Intact Protein and Middle-Up Measurements
Top-Down and Middle-Down Measurements | Bottom-Up Measurements
 
Reversed Phase - Introduction and Peptide Level Analysis

Everything you need to know about using reversed phase analysis for characterizing peptide fragments of biopharmaceuticals, including mAb digestion using trypsin, what a typical peptide may looks like, and which post-translational modifications can be identified and how.

Analysis at the Peptide Level | Common mAb Post-Translational Modifications
Glycoprofile

 
Reversed Phase - Peptide Mapping LC Conditions and Effects

This module will provide you with practical details of how to implement peptide mapping of biopharmaceuticals, from initial conditions to considering the effect of reversed phase parameters such as mobile phase additives, temperature, gradient slope, and column dimensions on the separation.

Typical Peptide Mapping Conditions | Mobile Phase Additives
Temperature | Gradient Slope/Time

 

Quick Guides


Reversed Phase HPLC for the Analysis of Biomolecules

Commonly reversed phase (RP), hydrophilic interaction (HILIC), ion exchange (IEX), and size exclusion (SEC) chromatographic modes will be combined to allow full characterization of complex biomolecules. The following article will introduce the fundamentals of biopharmaceutical analysis and cover the use of reversed phase HPLC in their analysis.

Reversed Phase HPLC for the Analysis of Biomolecules »

 
Hydrophobic Interaction Chromatography for Biopharmaceutical Analysis

Hydrophobic interaction chromatography (HIC) is a milder form of reversed phase liquid chromatography (RPLC).  Separation of analytes is based on hydrophobic interactions with the stationary phase; therefore, the elution order in HIC enables proteins to be ranked on the basis of their relative hydrophobicity.

HILIC for Biopharmaceutical Analysis »

 
Biopharmaceuticals Quiz

A little bit of fun to test your knowledge of biopharmaceuticals and their analysis.

Biopharmaceuticals Quiz »

 
HILIC, IEX, and SEC for the Analysis of Biomolecules

The enhanced complexity and variability that comes from the size of biopharmaceuticals, allied with the intricacy of the production process, mean chromatography is employed to a much greater extent during production and release testing. This article will examine the use of HILIC, IEX, and SEC for their full characterization.

HILIC, IEX, and SEC for the Analysis of Biomolecules »

 
Advances in Full Characterization of Protein Biopharmaceutical Post Translational Modifications

Post translational modification (PTMs) which are most characteristic in protein biopharmaceuticals include glycosylation (which comprises galactosylation, fucosylation, high mannose derivatives and sialylation), oxidation, phosphorylation, sulfation, lipidation, disulfide bond formation and deamidation, as well as (to a more limited extent) carboxylation, hydroxylation, and amidation; with many biopharmaceuticals having a combination of two or more PTMs.  PTMs need to be monitored during numerous stages of the manufacturing process and require identification, control of their levels, and assessment of their impact on the protein.  In order to do this many chromatographic (reversed phase and HILIC HPLC) and mass spectrometric (MS) techniques are employed.  This webcast will consider the types of modifications which can be assessed and what methods should be used to identify them, how various MS scanning techniques can be used for PTM analysis, as well as considering if the analysis mode itself can induce post translation modifications (collision induced dissociation vs. electron transfer dissociation (ETD) and electron capture dissociation (ECD)).  Finally, the use of LC-MS for protein determination of antibody-drug conjugates in biological matrices will be discussed.

Full Characterization of Protein Biopharmaceutical Post Translational Modifications »

 
Techniques Employed in Biopharmaceutical Analysis - Part I Reversed Phase and HILIC

This webcast shall interrogate the critical parameters required to successfully implement and optimize reversed phase analysis of biopharmaceuticals. We will also discuss the popularity behind glycosylated biopharmaceuticals, where heterogeneity arises in these molecules, and how glycans can be effectively liberated, characterized, and analyzed using HILIC.

Biopharmaceutical Analysis - Reversed Phase and HILIC »

 
HPLC Techniques in Biopharmaceutical Analysis

Biopharmaceuticals offer great hope in treating medical conditions which are currently poorly served at best by traditional pharmaceuticals.  It is estimated that there are over 400 biopharmaceuticals in clinical trials for in excess of 200 disease areas.  The enhanced complexity and variability that comes from the size of biopharmaceuticals, allied with the intricacy of the production process, mean chromatography is employed to a much greater extent during production and release testing.  This Essential Guide webcast will provide details of why differing modes of chromatography are required in the research, preparation and analysis of biopharmaceuticals and when they would be typically used.

HPLC Techniques in Biopharmaceutical Analysis »

 
 
 
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Dr. Anne Blackwell
Product Support Scientist
Agilent Technologies

Anne joined Agilent Technologies in 2012 after completing her PhD in mass spectrometry of non-covalent protein assemblies at the University of Arizona. 

She worked as an LC/MS applications chemist on metabolomics and ion mobility workflows until she joined the BioColumns Applications Team as a Product Support Scientist in 2016. 

Anne specializes in helping customers develop robust biomolecule separations, implement best practices to avoid common problems, and troubleshoot challenging problems when they arise.