Institute for International Research

Course Description

This course introduces fundamental concepts and teaches practical skills needed to establish small to large-scale transient protein production systems using mammalian cells. The class will examine in detail the four essential elements of any mammalian transient production system: cell lines, expression vectors, transient transfection and cell culture. You will learn the strengths and weaknesses of transient expression in order to make reasoned decisions about how and when to employ this rapid, cost-effective technique.

The course will help participants to understand differences and tradeoffs in producing recombinant proteins in HEK293, CHO, or other mammalian cells. We will also review expression vector basics and delve into advanced vector optimization, cloning strategies and large-scale preparation of plasmids. Specific focus will be placed on expression vector design for production of antibodies. The most commonly used transfection reagents and transfection methods will be examined, leading to discussions on optimization of large-scale transient transfection and overall expression system design matching transfection reagent to cells to culture medium.

Attendees will gain an understanding of the equipment needed to establish a transient production facility, methods to monitor culture conditions and how to assess transfection efficiency. The particular cell culture parameters and techniques that lead to maximal transient production will be explored and contrasted to culture strategies for production from stably engineered cell lines. A case study and in depth analysis of cell culture and transient transfection scale-up will be presented to give participants a working understanding of how to scale up production to benchtop bioreactors and beyond.

Complete Course Agenda

Overview and introduction to mammalian transient expression systems

• Rationale for transient protein production
• Comparison to non-mammalian production systems
• Comparison to stable expression systems
• The four fundamental elements of transient production

Cell Lines for transient production

• The HEK293 versus CHO decision
• Alternative mammalian cell lines
• Host cell differences in post-translational processing
• Adherent and suspension culture
• Engineered hosts for increased production

Expression plasmids

• Expression vector engineering
• Leading edge cloning strategies
• Coding sequence optimization
• Antibody expression vector strategies
• Plasmid production

Concepts in large-scale transient transfection

• Transfection reagents and methods
• Transfection optimization
• Transfection analysis and monitoring

Cell culture

• Culture medium for transient transfection
• Cell culture analysis and monitoring
• Optimizing culture conditions
• Culture supplements and enhancements for increased production

Equipment for transient protein production

• Small-scale culture systems
• Single use disposable bioreactors
• Benchtop and large-scale culture systems

Scale up

• Moving from flask to bioreactor
• 10 liter CHO case study

Additional Topics

• Cost considerations
• Downstream processes

Course Instructor

Henry C. Chiou, Ph.D., Technology Area Manager, Research and Development, Invitrogen Corporation
Dr. Henry Chiou has over 15 years of experience in mammalian expression technology and in nucleic acid delivery systems. He has broad expertise in expression vectors, cloning, cell biology and cell culture. Henry directs Invitrogen's R&D efforts in the development of new transfection reagents and transfection applications. He recently led the FreeStyle MAX program to produce a scaleable, suspension culture system for transient protein production from CHO and HEK293 cells. He frequently provides guidance or collaborates with various bioproduction, bioprocess or protein production Core facilities from biotech and pharma to help establish or optimize transient protein production systems. Dr Chiou obtained a bachelor's degree from Yale University in biochemistry and earned a doctorate in Molecular Pharmacology from Harvard University. He then completed a post-doctoral fellowship studying viral expression elements at the University of Pennsylvania. Prior to joining Invitrogen he worked for a number of years in small to mid-sized biotech companies developing biotherapeutics.