Using A Transient CHO System to Reach High Titer Expression Simply and Efficiently

By on October 27, 2015
Expert Session Summaries

We recently finished our Ask the Expert discussion on achieving gram per liter protein yields in a transient CHO system. During this Ask the Expert session, we discussed the advantages of a utilizing transient CHO system for protein production. Topics covered included expected yield, post translational modifications, consistency with stable cell lines, comparison to 293 transient systems. Specific cell culture related questions included, how to optimize transfection and some of the regularly mentioned strategies, i.e. temperature shifts, cell densities, growth factors, and DNA concentration.

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.

This Ask the Expert session was 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.

Below is a sneak peek of the discussion, for a full transcript, please see – Ask the Expert – achieving gram per liter protein yields in a transient CHO system.


What are the advantages of using a transient CHO system over a 293 system?

The Answer:

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 transient expression system, this is no longer the case, and indeed in many/most instances ExpiCHO is significantly higher expressing than Expi293. Secondly, since CHO cells are used to manufacture >70% of current licensed therapeutic proteins today, the ability to start drug development work in CHO cells and stay in CHO cells from discovery through bioproduction reduces risk, as transient CHO-derived proteins are more similar in terms of critical quality attributes to proteins made in stable CHO cells. Thirdly, the ExpiCHO expression system offers unparalleled flexibility in terms of protocol options. Three different protocols are provided in the ExpiCHO manual, Standard, High Titer and Max Titer protocols, which allow researchers to choose the option that best fits their needs in terms of equipment, time, number of steps and amount of protein desired. Additionally, because of the robustness of the ExpiCHO system, there are many ways that the system can be further modified to meet the particular needs of a given lab.


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?

The Answer:

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 bispecifics) and non-antibody proteins as well. One of the goals of the ExpiCHO system development was to allow for this seamless transition from discovery to pre-clinical studies using CHO-derived protein. Assuming that your protein expresses at reasonable titers, there should be no reason why this would not be possible. In many instances we have seen that proteins that express well in transient 293 express even better in ExpiCHO, with one study showing a nearly 3-fold improvement in titers across a panel of 20 mAbs, with every one of the mAbs expressing higher in ExpiCHO than in the Gold Standard Expi293 system. We have also seen numerous instances where a protein that does not express, or expresses very poorly, in 293 or other CHO systems, expresses well in the ExpiCHO system.


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?

The Answer:

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 shared from independent labs seems to suggest that if you were to run just one system to produce all of your proteins, that ExpiCHO would likely be the system to pick. Additionally, as you mention, the relevance of the protein generated in ExpiCHO to your production cell line is certainly also something that should be considered, as this allows for a more seamless workflow from discovery to clinic. Lastly, whether to keep a 293 system and a CHO system is based on the specific needs of your lab: for instance, if you are making protein reagents where human post-translational modifications are desired, then having a 293 system makes sense. If you are expressing only candidate therapeutics, then ExpiCHO would certainly be your first choice. Many/most labs that have the ability to run both systems are doing so, providing them with two options to best generate their proteins of interest in the most relevant cell line and providing the greatest likelihood that they will get the protein they need from either the Expi293 or the ExpiCHO system.

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