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The CHROMacademy Essential Guide Webcast:
Choosing the Correct Column and Mobile Phase for LC-MS Characterization of Biologics

Thursday 15th June 2017,
8:00am PST/ 11:00am EST/ 4:00pm BST/ 4:00pm CET

   
 

This webcast will focus on the parameters which are essential for the development of a successful RPLC-MS method for the characterization of a monoclonal antibody. 

The methods will allow for the analysis of the intact species, determination of absolute mass, examination of the major fragments, (Fab/Fc and Hc/Lc), and investigation of the small peptides produced by protease digestion.  

The importance of pore size and mobile phase additives will be addressed and guidance given on how to optimize methods for sensitivity and resolution.

Presented by

Dr. Andrew Coffey (Senior Applications Chemist, Agilent Technologies).

Topics Covered

  • Choosing the correct column pore size for mAb intact, fragment, and peptide analysis
  • Impact of column chemistry on selectivity and resolution
  • Impact of mobile phases additives on selectivity and sensitivity

Presenter Information »

 
 

LC-MS method development for biologics - Tutorial

This month’s webcast focused on developing LC-MS methods for the characterization of biologics.  Take your knowledge of biotherapeutic characterization to the next level by delving into our tutorial content.

Our Quick guides, CHROMacademy course content, and archived webcasts and tutorials have been selected to provide a well-rounded insight into all the chromatographic techniques used for the characterization of biologics.

Watch the webcast

eLearning Modules


 
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 | mAb peptide map | Common mAb PTMs

 
Reversed Phase - Peptide Mapping and 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.

Peptide mapping conditions | Mobile phase additives | Column length | Particle size and morphology

 
Reversed Phase - Protein Level Analysis and Optimization

Learn how to analyze intact proteins using reversed phase HPLC.  The effect of mobile phase additives, temperature, flow rate, and stationary phase chemistry are discussed.

Typical protein level conditions | Mobile phase additives | Gradient | Stationary phase

 

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 Analysis of Biomolecules »

 
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 »

 
Selecting Columns and Initial Conditions for IEX and SEC

Biopharmaceutical compounds are genetically engineered from living cells and have molecular weights in the range 2,000-2,000,000 Da with 10-2,000 reactive groups and are often comprised of a mixture of closely related variants; therefore, there is a wider degree of variability (a degree of heterogeneity) in comparison with small molecule pharmaceuticals. 

Columns and Initial Conditions for IEX and SEC »

 
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...

Protein Biopharmaceutical Post Translational Modifications »

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

Biopharmaceuticals offer great new possibilities and potential in treating various life threatening diseases such as cancer and autoimmune diseases. However, their complexity, resulting from their heterogeneity, represents a great challenge to the analytical chemist and a suite of techniques is required for their characterization and analysis. Reversed phase chromatography, the mainstay of traditional small molecule analysis, is employed at multiple biopharmaceutical levels due to its versatility, inherent relative efficiency and ease of hyphenation to a mass spectrometer...

Biopharmaceutical Analysis - Part I 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 trails 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. Andrew Coffey
Senior Applications Chemist
Agilent Technologies

Andy joined Polymer Laboratories in 1989 after completing his PhD in peptide synthesis at Wolverhampton, UK. He progressed from Senior Scientist to Product Manager looking after the HPLC column product line before focusing on the Solid Phase specialty particle businesses. He continued as Product Manager adding Biosolutions and Flash chromatography consumables product lines to his portfolio during the acquisition by Varian and then Agilent Technologies, and joined the Biocolumns Applications Team in August 2011.

Andy has considerable experience in reversed phase, ion-exchange and size exclusion liquid chromatography method development, technical support, and troubleshooting - and has a “hands on” attitude to problem solving!