The Analysis and Characterization of Protein Therapeutic Drugs
(Including Protein Structure and Function)
Instrumental Tools and
Techniques for the Characterization and Analysis of
Biotechnology-derived Polypeptide Therapeutics
In late 1995, the FDA initiated important changes in the regulation of protein therapeutic drugs which led to harmonization in the regulation of protein drugs and small molecule drugs. This change placed considerable emphasis on the analytical technologies used to characterize and analyze protein therapeutics. At the first “WCBP” Conference in January of 1997 a need was identified for a professional development short course focused on training scientists in just these analytical technologies. The resulting course was first presented in March of 1998 and has since been presented to over 2000 scientists worldwide. While undergoing many changes and revisions over the years, the course, now titled the Analysis and Characterization of Protein Therapeutic Drugs, has remained true to the original mission of training scientists in the current, state-of-the-art, technologies used to meet the challenges inherent in analyzing and characterization of complex biomolecules.
The Analysis and Characterization of Protein Therapeutic Drugs immerses those attending in the practical aspects of characterizing and analyzing therapeutic proteins and presents in an adsorbing manner the fundamental principles and practical uses of three fundamental analytical technologies. Topics extraneous to protein analysis are eliminated, allowing the course to focus on the points of each technology essential in protein characterization. Those attending leave with a clear sense of where each technology is used and how to most effectively implement each technology in developing and using effective, efficient analytical methods for characterizing and analyzing therapeutic proteins.
The Analysis and Characterization of Protein Therapeutics Drugs begins by providing a detailed description of protein structure and function followed by a complete discussion of protein modifications and degradations. It then teaches the essential principles and practices of three important instrumental analytical technologies as they relate to the analysis and characterization of polypeptides:
n Chromatography, especially reversed-phase HPLC
n Capillary Electrophoresis
n Mass Spectrometry
Morning: Protein Structure and Function. The class begins by outlining the various structural forms of proteins, describing the nature and functions of the natural amino acids, secondary, tertiary and quaternary structure. The role and purpose of tertiary structure in protein bioactivity is explained. Internet resources are mentioned.
Afternoon: Protein modifications and degradations discuss natural post-translational modifications, degradations and engineered modifications to therapeutic proteins. The class will include a description of protein degradations such as deamidation, oxidation, denaturation and aggregation, post-translational modifications such as glycosylation and disulfide bonds and engineered modifications such as pegylation and drug conjugates. Peptide mapping is described as a foundation to discuss the analytical technologies. Emphasis is placed on determining and defining Critical Quality Attributes and how to monitor these.
Morning: Reversed-phase High Performance Liquid Chromatography.
· Basic theory and mechanism of polypeptide separations.
· Developing an analytical method for the analysis of polypeptides. Deciding on the right column. Selecting the elution solvent including the organic modifier, the ion-pairing reagent, the pH, the flow rate and the gradient conditions. Detection mode. The effect of Temperature.
· Developing a reversed-phase HPLC assay. The steps in developing a robust analytical method.
· Tips for the effective use of reversed-phase HPLC in polypeptide separations.
· Examples of how reversed-phase HPLC is used to characterize and analyze protein degradations, post-translational modifications and other important protein attributes.
Afternoon: Other forms of chromatography used in polypeptide analysis and in glycan analysis.
· Ion Exchange Chromatography (Charge variants)
· Hydrophobic interaction chromatography (Protein modifications)
· Size exclusion chromatography (Aggregation and Pegylation)
· Chromatography in glycan analysis: High pH Anion Exchange, Normal phase and HILIC.
Morning: Capillary Electrophoresis
§ Basic principles of CE. What is CE? Comparison with HPLC and gel electrophoresis. Understanding basic terms in CE: Mobility, Migration, Separation efficiency, and Electroosmotic flow. Basic CE instrumentation. Sample detection. Sample injection. The capillary and temperature control.
§ Capillary Zone Electrophoresis. What is capillary zone electrophoresis? Developing a CZE method including selecting the capillary, choosing the best buffers, the importance of pH, the role of the sample matrix, the use of buffer additives and choosing the correct voltage. Sample stacking, what it is and how to use it.
§ Capillary Isoelectric focusing. A description of Capillary Isoelectric focusing. The steps in CIEF. Practical use of CIEF
§ Capillary Replacable Gel Electrophoresis (CE-SDS). A brief discussion of CE-SDSand how it is used in bioanalytical separations.
§ How capillary electrophoresis is used to meet the needs and challenges of polypeptide characterization and analysis.
Afternoon: Mass Spectrometry
· Basic principles and terminology of mass spectrometry
· Ionization sources: Matrix-Assisted Laser Desorption Ionization (MALDI). Electrospray. For each ionization source, a discussion of selecting conditions, the affect of concomitant species and optimizing performance.
· Mass analyzers: Quadrupole. Ion Trap. Time-of-Flight. Orbitrap
Morning: Mass Spectrometry (continued from Day 3.)
· Interfacing mass spectrometry to HPLC and CE
· Fragmentation of ions in mass spectrometry. Collisionally-Induced Dissociation (CID). In-source CID in electrospray.
· Applications. How mass spectrometry is used to meet the needs and challenges in the characterization and analysis of protein therapeutic drugs. Determination of molecular weight of proteins by electrospray-MS. The role of mass spectrometry in peptide maps. Sequencing peptides by electrospray CID MS and by MALDI-MS. How mass spectrometry is used to determine protein modifications and degradations.
· The role of mass spectrometry in glycan analysis.
Note: Some topics will overlap into the following time period due to class discussion and class flow.
n "Very informative and comprehensive course. I enjoyed it!"
n ''Remarkable amount of resource information given with speakers experience made for easy adsorption of in-depth technical information."
n "A thorough crash course for someone with small molecule experience beginning research on biomolecules."
n '"I learned more theoretical and practical analytical methods information in these three days than in one year at school or work!"
n This course teaches the basic principles of the important instrumental analytical technologies used in polypeptide analysis and characterization and it focuses on how these are used to determine polypeptide characteristics, post-translational modifications and protein degradation products. In this course you will learn:
n What can happen to a protein therapeutic and how to show that a protein is unchanged.
n What are the important parameters in RP-HPLC analysis of polypeptides and how these affect the analytical method.
n How RP-HPLC can best be used to analyze and characterize therapeutic proteins.
n When and how to use capillary electrophoresis for monitoring protein therapeutics.
n How to set the analytical parameters for a CE analysis.
n What is the role of mass spectrometry in protein therapeutic analysis and what is its future.
n How to use mass spectrometry to analyze protein therapeutics.
n How protein databases are used for protein identification.
n What is glycosylation and how is it determined and measured.
n Scientists newly involved in the analysis or characterization of biotechnology-derived polypeptide pharmaceutical products.
n Biochemists and protein chemists seeking to understand the instrumental techniques used to characterize and analyze polypeptides.
n Anyone who would like to be immersed in an adsorbing, and sometimes entertaining, course on the analysis of protein therapeutics.
Instructor. C. David Carr. A technical short course is only as good as its instructor. David has over thirty-five years experience in the analytical technology field and is an expert in High Performance Liquid Chromatography. He is very familiar with capillary electrophoresis and mass spectrometry as applied to the analysis of polypeptides. Dave has provided instruction on HPLC and the use of HPLC in the analysis and characterization of polypeptides for many years. He has a facility for explaining the concepts and practices involved in the analysis of polypeptides in a manner that makes them clear and easy to understand. Course attendees can anticipate a very educational, yet entertaining, presentation with ample opportunity for discussion and questions
Technical Advisory Board
Dr. Robert Garnick, formerly Vice President of Quality, Genentech.
Dr. William Hancock, former President of the California Separation Science Society and past co-chair of the Well-Characterized Biotechnology Conference. Formerly with Genentech and ThermoFinnigan, Dr. Hancock is currently with the Barnett Research Institute at Northeastern University.
Dr. Alan Herman, formerly Director of Analytical Research and Development for Amgen.
Dr. Ron Orlando, faculty member of the Complex Carbohydrate Research Center in Athens, Georgia
Schedule and Registration
The Analysis and Characterization of Protein Therapeutic Drugs (Including Protein Structure and Function)
October 15 - 18, 2013 South San Francisco, California
Note: it is possible to attend the first day only for anyone desiring a fundamental course in protein structure and function but not interested in the detailed discussion of analytical technologies. Contact us for more information.
To register for the course, click REGISTRATION
This course is also available to be presented at your company as an in-house course. Classes can be optimized for specific company needs.
By mail: P.O. Box 952 Wrightwood California 92397