Achieve gram per liter protein yields in a transient CHO system


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.

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Question 1

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 (ExpiCHO-S cells), a new high-density growth and expression medium with matched feed as well as a high-efficiency transfection reagent and expression enhancer, all of which work together synergistically to maximize protein titers. ExpiCHO is capable of expressing many proteins at significantly higher levels than Expi293, with maximum titers up to 3g/L for human IgG compared to 1g/L in Expi293. Additionally, many proteins that express at very low levels, or not at all, in 293 cells can be expressed in ExpiCHO with excellent results.

Question 2

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

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.

Question 3

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 into pre-clinical and finally clinical trials. One of the main goals during the development of the ExpiCHO transient expression system was to ensure a more seamless transition from early drug discovery through clinical production allowing for researchers to start their work in CHO and stay in CHO throughout the entire drug development process. To this end, it is well documented that post-translational modifications in 293 cells and CHO cells are distinct from one another, with glycosylation patterns and sialic acid content being two instances of differences between the cells types. The ExpiCHO expression system demonstrated glycosylation patterns for a human IgG that were highly comparable to those of the same antibody expressed stably in CHO-S cells; in contrast, the glycan patterns for the same antibody expressed in 293 cells were strikingly different, with significant differences observed in the percentages of G0F, G1F and G2F glycoforms compared to CHO-derived material. The maintenance of protein critical quality attributes is critical in ensuring similar bioactivity and pharmacokinetic profiles of therapeutic proteins from pre-clinical to clinical trials.

Question 4

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 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.

Question 5

How much optimization is required with your system and do you use serum?

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 the past, then you already have all of the equipment that you need to run ExpiCHO and the level of training necessary to run the ExpiCHO system would be minimal/none. The ExpiCHO system is designed to work straight out of the box so the only optimization that would need to be done would be to simply ensure that your particular lab equipment is being used within the guidelines provided in the ExpiCHO protocol.

Question 6

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 aggregate prone during cellular processing. However, simply adding a temperature shift to an existing protocol without simultaneous optimization of other aspects of the protocol may provide modest gains, at best. As part of the ExpiCHO expression system, we provide three different protocols: Standard titer protocol which is performed at 37C as well as High Titer and Max Titer protocols that utilize a temperature shift to 32C. In this case, we have rigorously optimized the three protocols to ensure that the temperature shift is providing benefit to most proteins. For example, for a given IgG that expresses at 1g/L in the Standard protocol at 37C, we have seen this same antibody express at 2g/L in the High Titer protocol at 32C and at 3g/L in the Max Titer protocol at 32C. However, we, and others, have also seen that some proteins express better in the Standard protocol in the ExpiCHO system, again indicating that there is some protein dependence in which protocol/temperature works best for a given protein. The ability to choose temperature shift or no temperature shift in the ExpiCHO system provides two different options to best express your protein of interest.

Question 7

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 expressing CHO clone, ExpiCHO-S cells, a new expression media and feed, as well as a new high-efficiency transfection reagent and expression enhancers, all of which when optimized to work together increased protein titers of a human IgG from 20mg/L in the FreeStyleCHO transient system to 3000mg/L in the ExpiCHO system. It is truly through the synergistic interaction of all of the components of the system where these log-fold improvements in protein titers can be observed.

Question 8

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 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.

Question 9

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 as high as 3g/L. ExpiCHO is a fully optimized CHO-based transient expression system that expresses at levels previously seen only in stable CHO cultures.

Question 10

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 1x10^6 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 transfection reagent itself as well as how well the culture media can maintain the high level of cell health at higher densities needed for high efficiency transfection, 2) at high transfection densities, cells will rapidly overgrow the cultures post transfection and unless your culture media can handle this, the culture will crash and productivity will be poor. The ExpiCHO transient expression system is designed to deal with all of these problems and has been developed to transfect CHO cells at 6x10^6 cells/mL, or even higher, densities significantly higher than the standard 1x10^6 cells/mL transfections that are typical. ExpiCHO utilizes this high cell density to increase the number of cells expressing your protein and to ensure maximal yields.

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