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
Course Schedule
Class begins each day at 8:30 am and ends about 4:00 pm each day, except
for the fourth day, when class ends at noon.
Day 1
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.
Day 2
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.
Day
3
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
Day
4
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.
§
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.
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.
Bioanalytical Technologies
Telephone: 760-409-0871
Facsimile: 760-281-5926
e-mail: davidc.carr@verizon.net
By mail: P.O. Box 952 Wrightwood California
92397