- Cool Tool – The Human Protein AtlasPosted 6 days ago
- Optimization of Roche Liberase MNP-S GMP Grade in the Enzymatic Digestion of Human Umbilical Cord for the Isolation of Mesenchymal Stem CellsPosted 7 days ago
- Ask the Expert – Maximizing Transient Protein ProductionPosted 1 week ago
- Enabling Viral Vector Production and Vaccine Manufacturing using the iCELLis – a single-use, automated, and closed manufacturing platformPosted 2 weeks ago
- Cell Culture Basics – Mycoplasma 101 – A practical guide to prevention, detection and elimination of mycoplasma contaminationPosted 3 weeks ago
- Going to BPI West 2017? Don’t miss these great talks and activities!Posted 4 weeks ago
- Cool Tool – PRIME-XV® T Cell CDM – First Commercially Available Chemically-defined, Animal-component-free Medium for T Cell CulturePosted 4 weeks ago
- Increasing Protein Production with Novel Cell-Ess Titer Boost without Affecting the Metabolic ProfilePosted 1 month ago
- Continuous Processing Optimization with Smarter ToolsPosted 1 month ago
- Cool Tool – Generation of Neural Stem Cells from AlphaSTEM Cultured Pluripotent Stem CellsPosted 1 month ago
Tools and Strategies for Transient Protein Production in Mammalian Cells – A Discussion
We recently finished our Ask the Expert discussion on Transient Protein Production in Mammalian Cells. This week we had several interesting questions and helpful tips on topics including best practices, optimal complexation, best cell densities, achieving high volumes and expressing intercellular proteins. We also covered an overview of transient systems, transient expression media, and increasing expression yields.
This Ask the Expert Session was sponsored by Thermo Fisher Scientific and hosted by Jonathan Zmuda, Ph.D. Dr. Zmuda is the Associate Director of Cell Biology in the Life Sciences Solutions Group at Thermo Fisher Scientific, Inc. Prominent among his roles, Dr. Zmuda leads a team dedicated to discovering and developing new technologies and products useful for cell biology applications including protein expression, cell culture, rare cell analysis and instrumentation while also focusing on the development, qualification and validation of cell-based assays used for QC testing. Dr. Zmuda received his Ph.D. in Cell Biology from the University of Maryland, College Park and his undergraduate degree from Dickinson College in Carlisle, PA.
Scientists interested in setting up a rapid and robust mammalian expression process to produce milligram to gram quantities of recombinant protein should visit this Ask the Expert session. Below is a sneak peek, for a full transcript of the discussion, please see – Ask the Expert – Transient Protein Production in Mammalian Cells.
We have used transient production for a few projects, but are now looking at expanding our use. What kind of volumes could we reasonably expect if we scaled up and would you recommend HEK or CHO cells for high volumes? Also, if we optimized our system, what is the fastest timeline could we achieve?
Many researchers routinely use HEK293 cells at 1L scales in 3L shake flasks and at even larger volumes in 10L or 20L Wave bags or benchtop bioreactors. Certainly CHO cells can also be scaled up, as the vast majority of biotherapuetics are made in CHO cells at multi thousand-liter scales. Scaling up transient systems from mLs to liters is not trivial, and some cells will scale up better than others. When scaling up the dynamics in the shake flasks can vary considerably and one has to consider shake speeds, gas exchange, baffled vs. non-baffled flasks and temperature among other variables. At this time, I would recommend the use of HEK293 cells, as the yields from these cells tend to be considerably higher than CHO. Once again, the Expi293 system has been validated to generated consistent titers from multiwell plates all the way up to 10L Wave bags.
I am using a rather basic medium for my HEK293 cells and have seen mediocre transfection efficiency. I am wondering if I could get an increase in efficiency by simply using a richer media. If so, are there supplements you would recommend?
It is likely not simply an issue of using a “richer” media, but more so a better (i.e. possibly different) media that allows for more rapid growth of your cells so that the highest percentage of cells as possible are actively dividing at the time of transfection to promote DNA entry into the nucleus. There are many media developed specifically to support rapid cell growth and high density cultures and I would suggest screening some of the various 293 media available to find the one that works best for you rather than simply adding feeds or other supplements to your existing media. Gibco FreeStyle293 media and the newer Expi293 media are specifically designed to support high density HEK293 growth and transfection.
I have read that pre-treating cells with DMSO can lead to higher yields, do you find this is true. Is there another yield raising method that does not involve DMSO?
The use of DMSO to enhance protein titers has been widely reported in the literature, however, we do not employ this method due to the toxic nature of DMSO as well as the undefined nature of its effects on cells. For certain cell lines, it may be advantageous to reduce the temperature of the cultures from 37C to 32C following transfection to shift the cell’s usage of energy from replication to protein production. Other strategies include the use of compounds such as sodium butyrate to slow cell growth. As always, some of the best ways to improve protein yields are by ensuring that you have healthy cells and a fully optimized protocol encompassing the growth and maintenance of the cells, the DNA complexation reaction and the transfection of the cells themselves. Ensuring that your cells are happy and healthy goes a long way towards ensuring maximal protein titers.