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The Analysis and Characterization of Protein Therapeutic Drugs
Instrumental Tools, Techniques and Tips for the Characterization and Analysis of
Biotechnology-derived Polypeptide Therapeutics

New regulations issued by the FDA on May 14, 1996 substantially changed the way in which biotechnology-derived pharmaceuticals are regulated, placing greater emphasis on the methodologies used to analyze biotechnology-derived polypeptide therapeutics. Fundamentals in Analytical Biotechnology 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 these three analytical technologies. Topics extraneous to protein analysis are eliminated, allowing the course to focus on the points of each technology essential in protein characterization. The materials can become almost overwhelming because of the sheer weight of information presented, and yet all points become clear because of the structure of the class and the ability of the instructor to present each topic clearly. 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 is a condensed and focused course of instruction on the instrumental analysis and characterization of polypeptides, emphasizing three important instrumental analytical technologies as they relate to the analysis and characterization of polypeptides.

n Protein Structure and Function. Modifications to proteins that may affect function or potency.

n Chromatography, including reversed-phase HPLC, ion exchange and other techniques related to protein analysis

n Capillary Electrophoresis, a technique used for the analysis of charge variants, antibodies and glycosylation

n Mass Spectrometry, a powerful technique for the analysis and characterization of protein modifications.


Course Outline

This is a three and a half day classroom lecture course focusing on the structure and function of proteins, modifications to proteins that may affect their function, and the fundamental principles and practical use of three major instrumental techniques Chromatography, especially reversed-phase HPLC (other chromatography techniques are also discussed), capillary electrophoresis and mass spectrometry - in the analysis and characterization of biotechnology-derived polypeptide biopharmaceuticals.

Class begins at 8:30am each day and concludes between 3 and 4pm. On Day 4, class concludes at 12:00 noon.


Day 1

Introduction to the course including a discussion of the history of the regulation of protein therapeutics.

Protein Structure and Function

n  Amino acids. Structure and character.

n  The peptide bond

n  The nature of the side-chains of the common amino acids.

n  The interactions which form and stabilize polypeptide

n  Secondary structure. The alpha-helix and beta-sheets

n  Protein tertiary structure and its role in biological activity

n  How primary and secondary structure lead to tertiary structure.

n  Protein quaternary structure

n  Why structure matters.


Protein Degradations and Post-translational Modifications

n  Post-translational modifications such as glycosylation, the addition of oligosaccharides to polypeptides.

n  Deamidation and oxidation and their effects on protein function and biological activity .protein stability.

n  Changes to tertiary structure. Denaturation and aggregation.

n  Engineered proteins. Protein conjugates. Monoclonal antibody conjugates. Pegylation.

Day 2

Reversed-phase High Performance Liquid Chromatography.

n  Basic theory and mechanism of polypeptide separations.

n  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 affect of Temperature.

n  Developing a reversed-phase HPLC assay. The steps in developing a robust analytical method.

n  Tips for the effective use of reversed-phase HPLC in polypeptide separations.

n  Examples of how reversed-phase HPLC is used to characterize and analyze protein degradations, post-translational modifications and other important protein attributes.


Other types of chromatography in polypeptide analysis.

n  Ion Exchange Chromatography (Charge variants)

n  Hydrophobic interaction chromatography (Protein modifications)

n  Size exclusion chromatography (Aggregation and Pegylation)

n  Chromatography used in the analysis of glycosylation: High pH Anion Exchange, Normal phase and HILIC.


Day 3

Capillary Electrophoresis

n  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.

n  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.

n  Capillary Isoelectric focusing. A description of Capillary Isoelectric focusing. The steps in CIEF. Practical use of CIEF

n  Capillary Replacable Gel Electrophoresis (CE-SDS). A brief discussion of CE-SDSand how it is used in bioanalytical separations.

n  How capillary electrophoresis is used to meet the needs and challenges of polypeptide characterization and analysis.

Mass Spectrometry

n  Basic principles and terminology of mass spectrometry

n  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.

n  Mass analyzers: Quadrupole. Ion Trap. Time-of-Flight. Orbitrap

n  Interfacing mass spectrometry to HPLC and CE



Day 4. Mass spectrometry continued

n  Fragmentation of ions in mass spectrometry. Collisionally-Induced Dissociation (CID). In-source CID in electrospray.

n  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.

n  The role of mass spectrometry in glycan analysis.


What previous attendees have said about The Analysis and Characterization of Protein Therapeutic Drugs

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!"

What You Will Learn.

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.

Who will benefit from this course?

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.

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