How to Improve Protein Productivity Through Scale Up, Including Single-Use Bioreactors


Enhancing protein production is a common goal in the biomanufacturing industry. However, scalability is a problem faced across many platforms in manufacturing.  A desired outcome observed in shake flasks, such as increased protein productivity, is not always observed in large scale bioreactors.  Further, the move toward single-use bioreactors has posed an additional challenge, as productivity improvements observed in glass shake flasks or stainless steel bioreactors may not be observed in plastic bioreactor bags.  Availability of lipids in media is one factor to consider during bioproduction scale-up and when moving toward single-use system.

During this Ask the Expert session, we will be discussing considerations to be made regarding bioproduction scale-up and single-use bioreactors. Essential Pharmaceuticals is addressing these challenges with their Cell-Ess lipid supplement.  Cell-Ess was used as a feed at 5% (v/v) in a small Wave bioreactor resulting in greater than 25% monoclonal antibody titer increase, which was similar to the gains seen in earlier experiments using shake flasks.  Small bioreactors have been used as models to show scalability to up to 10,000-liter manufacturing runs.  These new data suggest supplementing with Cell-Ess as a feed will result in increased monoclonal antibody titers for 10,000-liter bioreactor runs, including in single-use bioreactors.

Who should visit?

  • Those in early development of bioproduction of proteins in eukaryotic cells
  • Those in media optimization or yield optimization for scaled bioproduction of proteins
  • Department Directors, Program Directors, Project leaders, involved in optimization of large scale bioproduction of proteins.

Types of Questions?

  • How does lipid availability in media influence protein productivity?
  • What can contribute to differences observed during the scale up process?
  • Why might lipid availability be different in single-use bioreactors?
  • What mitigations can improve lipid half-life and availability in media?
  • How can supplements be used in large scale bioproduction to improve protein productivity?

This Ask the Expert session is sponsored by Essential Pharmaceuticals and is hosted by Dr. Adam Elhofy, Ph.D., CSO. Dr. Elhofy developed the core technology for the Ess line of products and aided in creating patents around novel uses of materials.  Dr. Elhofy has over 14 years of scientific research experience in the areas of immunology, neuroscience, and oncology.  He was funded by both the National Institutes of Health and the Multiple Sclerosis society as an investigator at Northwestern University Medical School. His doctoral research won him the award of the Top 5 trainee scientists by the American Association of Immunologists. Dr. Elhofy has 14 scientific publications in peer reviewed journals. He has played a variety of roles with start-up biotech companies ranging from Principal Investigator to Director of Corporate Development.

For more information, please see “New Advances Pose New Challenges to Bioproduction.”

Question 1

Do you know which CHO cell lines are more impacted by the addition of lipids in the media?

Cell-Ess has been tested on several CHO cell lines. The effect has been similar across the different genetic backgrounds. The amount of Cell-Ess used varies more based on the SFM used to grow the CHO cells. Once an optimized stragtegy is reached, the increase in titer is typically and reproducibly in the 20%-30% range.

Question 2

We are using HEK cells for our smaller scale protein production. Have you tried this supplement in HEK?

We are currently trying the product with HEK cells grown in a SF media. We are eagerly awaiting the results.

Question 3

Can this be used for MDCK cells? We are culturing cells in a WAVE bioreactor, and they could use some help with growth and viability.

Cell-Ess has been used with MDCK cells that were adapted from serum. We have not tried Cell-Ess as a supplement to MDCK cells grown in an SFM. We anticipate there would also be a benefit based on the results of the serum adapted CHO cells.

Question 4

We work with CHO transfection pools for protein production. We typically use a more complex media for this application. Have you seen your product can be helpful with transfection pools?

We have seen a benefit in CHO cell pools. As with isolated clones used for production, the base SFM drives the amount of Cell-Ess needed for an observed benefit. The typical time for pools grown is shorter than that for production runs so most likely an initial supplement tends to be sufficient. We can work with you to give guidelines based on our experience what will work best for your specific goals.

Question 5

I am curious, we are getting good productivity in our glass benchtop bioreactors, but when we scale up to 200 liters, our productivity drops. We have seen that this is due to a decrease in vcd. What could you recommend we try?

We have seen similar results in glass, steel, and SUBs with Cell-Ess. I have several questions and maybe we can go through them together offline. For example what does your VCD curve look like in each vessel? Do you know what may account for the different curve shapes? There are several other questions we can go through. Please feel free to use Essential as a resource.

Question 6

Is this product removed through normal downstream purification processes, i.e. protein A?

This product does not affect the downstream process. Cell-Ess does not bind to or prevent the binding of mAb to protein A.

Question 7

Have you looked at the effect of your product on glycosylation profile or charge variants? We are trying to match an existing product profile.

We have looked at glycosylation. We do not significantly change the glycosylation pattern. In fact we have preliminary evidence to suggest Cell-Ess may create an environment for more reproducible glycosylation profiles.

Question 8

When would you add this is it a supplement or a feed?

Cell-Ess is can be both a supplement and a feed. The nomenclature for the manufacturing process can be a little confusing. For example, supplements can be used as feeds. What we found is that each clone is different, and the media requirement may be slightly different. Therefore, optimization is the key. In some cases, Cell-Ess is best as a one-time use at time zero. Other times Cell-Ess is best used as 2 or even 3 feeds. The important thing is that once an optimal point is reached, then it stays the same, and the reproducibility is observed for run after run.

Question 9

We are adding simple fatty acids to our media, and we use plastic bioreactors. Are our simple fatty acids sticking to the plastic, or is it just complex fatty acids that are the problem?

Free fatty acids are added in some media optimization strategies. Free fatty acids have a very short half-life once they are in solution. Furthermore, all types of free fatty acids (both simple and complex) bind to vessel walls, especially in SUBs, where they will be mostly adhere to the vessel with 24 hours.

Question 10

Each time we add lipids to our media, we must make fresh solution and titrate the appropriate amount to add to avoid cell death. Do we have to undergo the same process with your supplement?

Cell-Ess is stable in solution. There are issues with free fatty acid and free cholesterol with stability and solubility. In addition, the carrier may cause a problem. All of these issues have to be monitored and accounted for in cultures lasting over 4 days. With longer cultures, media depletion becomes a concern, and feeds are used to supplement the culture. For free fatty acids, free cholesterol, or cholesterol with a carrier in extended cultures, toxicity becomes a concern with the second or third feed and can be variable from run to run based on the break down or initial stability of the product. Cell-Ess deploys a novel method of delivery so these are not issues when optimized with Cell-Ess.

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