CHO cells are the predominant host for biotherapeutic protein expression, with roughly 70% of licensed biologics manufactured in CHO. Multiple attributes make CHO cells desirable for bioproduction including the ability to adapt to high-density suspension culture in serum-free and chemically-defined media and the incorporation of post-translational modifications that are biologically-active in humans. For these reasons, the ability to produce transient CHO-derived proteins early on during drug development is highly advantageous to minimize, as much as possible, changes in protein quality/function observed when moving from R&D to bioproduction. Unfortunately, CHO cells express lower levels of protein than HEK293 cells in existing transient systems, in some instances 50-100 times less than the best 293-based systems. The recent introduction of the ExpiCHO transient expression system allows for unprecedented access to CHO-derived proteins early on during candidate selection and may serve to revolutionize the use of CHO cells for transient protein expression during the drug development process.
Who should visit the session?
- Do you need a high-expressing transient CHO expression system for your work?
- Do you use 293 cells for drug candidate transient expression due to low levels of protein produced in transient CHO systems, even though your drug candidates will eventually be produced in CHO cells?
- Are post-translational modifications of proteins critical to your work?
- Do you encounter hard to express proteins that do not express well in 293 cells and would like another option to improve your protein titers?
This Ask the Expert session is sponsored by Thermo Fisher Scientific, Inc and is hosted by Jonathan Zmuda, Ph.D., Associate Director of Cell Biology in the Life Sciences Solutions Group. 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. 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.
please take advantage of the opportunity to ask our expert a question and participate in a lively discussion on transient CHO expression.
We are not seeing the results we would like in 293 cells and need a transient CHO system. What would you recommend?
Thermo Fisher Scientific recently launched a high-expressing, all-in-one CHO-based transient expression system, ExpiCHO™, which builds on the knowledge of the Gold Standard Expi293™ expression system that has been widely used by researchers and drug development scientists for many years. The ExpiCHO expression system was developed from the ground up incorporating a new, high-expressing CHO clone … Continued
In the past, 293 cells were the overwhelming choice of researchers performing transient protein expression because 293 cells could generate significantly higher titers than CHO cells in a transient setting. For example, Expi293 is capable of generating titers greater than 50-fold higher than the FreeStyle CHO™ transient expression system. With the introduction of the ExpiCHO … Continued
We need post-translational modifications of our transiently expressed proteins that are similar to those of our stably expressed proteins to be used for clinical trials. How can a transient CHO system help?
In addition to the high titers and the ability of the ExpiCHO system to express proteins that don’t express well, or at all, in 293 cells, the key reason to use CHO cells for transient protein expression is to reduce the risk of critical quality attributes of your protein changing when transitioning from early discovery … Continued
We are having some difficulties with expression of one of our proteins in 293. We are considering trying CHO instead and possibly changing to a CHO platform for consistency with our manufacturing cell line. Is there any reason that you would recommend keeping both 293 and CHO as transient expression systems or would adopting a CHO platform be fine?
We have seen multiple instances where a protein that does not express in Expi293, or expresses at a very low level, is expressed well in the ExpiCHO system. At this time, there is not a consensus understanding as to which proteins express better in 293 cells vs. CHO cells, however, our experiences and some data … Continued
There is no serum used in the system. In fact, both the ExpiCHO medium and ExpiCHO Feed are chemically defined, protein-free, serum-free, animal-origin free formulation produced under GMP guidelines. The amount of optimization of the system depends upon the experience your lab has with transient protein expression. If you have used the Expi293 system in … Continued
We have been investigating temperature shifts but we haven’t seen much improvement using this technique. Any thoughts?
The answer depends on the desired outcome that you are looking to get from the temperature shift: to increase titers, to improve the quality of a given protein or both. Temp shifts are typically used for both increasing titers as well as for assisting in the expression of hard-to-express proteins, such as proteins that are … Continued
Using CHO for transient expression has not been giving use the productivity we are looking for. Do you think adding lipids or growth factors like insulin or IGF would be helpful?
The optimization of single factors in a CHO-based transient expression system provides modest gains, at best. In many instances existing transient CHO systems express at 10-50mg/L titers, such that even a 50% increase in titer is not a significant improvement. The ExpiCHO system has been developed from the ground up by identification of a high … Continued
Our group has been tasked with producing a fairly high amount of protein for pre-clinical study. We’d rather not take the time and just use transient expression instead. What is the upper limit in terms of production? Range of expected titer?
For a high-expressing human IgG, pre-purification titers of up to 3g/L have been obtained in the ExpiCHO expression system, titers previously only attained in stable cell lines. For many other antibodies, ranges from 100’s mg/L to multiple grams/L have been generated in external laboratories. Similarly, the ExpiCHO system does an excellent job expressing antibody-like (ie … Continued
I routinely see in literature transient cho expression at around 1g/l. However I can’t get above 40 mg/L. I’ve tried a bunch of different commercial transfection reagents, temperature shifts, densities, dna concentrations, etc. Do you have any thoughts of what else I could try?
Actually, and unfortunately, 40mg/L is not terribly uncommon for most transient CHO systems. Reports of 1g/L in CHO tend to be for stable cell lines, not traditional transient systems. The short answer to your question is to try the ExpiCHO expression system. Many proteins have been expressed at >1g/L in the ExpiCHO system, with titers … Continued
We are having trouble determining the optimal cell density for our transient expression in CHO. Any suggestions?
The optimal cell density is dependent upon a number of factors, but in a typical system comprising cells, media and transfection reagent, most often 1×10^6 cells/mL is the cell density chosen for a number of reasons: 1) Typical transfection reagents do not work well at very high cell densities—this is both a consequence of the … Continued