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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 shear 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.
Ø
Chromatography, especially reversed-phase HPLC
Ø
Capillary Electrophoresis
Ø
Mass Spectrometry
Course Outline
This is a three day classroom lecture course focusing
on the fundamental principles and practical use of three major instrumental
techniques – Chromatography, especially reversed-phase HPLC, capillary
electrophoresis and mass spectrometry - in the analysis and characterization of
biotechnology-derived polypeptide biopharmaceuticals. Other chromatography
techniques are also discussed.
Day 1
Introduction to the course including a discussion of the history of the regulation of
protein therapeutics focusing on recent changes eliminating the establishment
license requirement and adoption of a Biologics Application.
Needs and challenges in the analysis of biotechnology-derived therapeutics will
include a description of protein degradations such as deamidation, oxidation
and aggregation, post-translational modifications such as glycosylation and
peptide mapping as a foundation to discuss the analytical technologies.
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. The affect of
system hardware.
n Developing a reversed-phase 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.
Day 2
Other types of
chromatography in polypeptide analysis.
n Ion Exchange Chromatography (Charge variants)
n High
pH Anion Exchange Chromatography (Glycan Analysis)
n Hydrophobic interaction chromatography
(Protein modifications)
n Size exclusion chromatography (Aggregation and Pegylation)
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.
Day 3
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
n Interfacing mass spectrometry
to HPLC and CE
n Fragmentation of ions in mass spectrometry. Collisionally-Induced Dissociation (CID). In-source CID in
electrospray. Post-source decay in MALDI
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, 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