Improving Glycosylation Patterns and Consistency Through Media Optimization

We recently finished our Ask the Expert discussion, “Media Optimization Can Improve Glycosylation Patterns and Consistency to Impact Protein Efficacy”. During this Ask the Expert session, we discussed factors that influence glycosylation, the relationship between media and glycosylation, and the effect of glycosylation on the protein. Additional topics included glycosylation in biosimilars, glycoengineering, other post-translational modifications and protein aggregation.

When producing a biologic therapeutic, protein quality is a key consideration. While manufacturing parameters such as high protein yield, protein solubility and protein stability are important, it is also critical that the biologic exhibits the desired therapeutic activity. Post-translational modifications impact efficacy and pharmacokinetics of the biotherapeutic, where improper glycosylation can result in an ineffective drug. Proteins are commonly post-translationally modified by adding sugar residues in a process called glycosylation. Half of all human proteins are estimated to be glycosylated.

Essential Pharmaceuticals is addressing the challenges associated with glycosylation including increasing consistency of glycosylation profiles with their Cell-Ess universal titer boost and optimizer. Cell-Ess was used as a feed in a Wave bioreactor, resulting in greater than 25% increase in monoclonal antibody titer, which was similar to the titer increases observed in earlier experiments using shake flasks. In addition to increasing titer, Cell-Ess was shown to significantly increase consistency of glycosylation patterns when used as a feed. Further, Cell-Ess was shown to increase higher order glycoforms measured by increase in galactosylation. These new data suggest supplementing with Cell-Ess as a feed increases protein titer while improving or maintaining quality. To learn more about the use of Cell-Ess, please see our previous article, Cool Tool – Novel universal titer boost and enhancer improves CHO cell protein production in small bioreactors.

This Ask the Expert session was 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 15 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.

Below is a sneak peek of the discussion, for a full transcript, please see – Ask the Expert – Media Optimization Can Improve Glycosylation Patterns and Consistency to Impact Protein Efficacy.


What factors influence glycosylation?

The Answer:

Glycosylation is affected by several factors, including: movement of the protein through the ER and Golgi apparatus, environmental factors, and sugar availability.  The movement of protein through ER and Golgi apparatus can be affected by cholesterol and other free fatty acid availability that impact health of ER and Golgi membranes.  Environmental factors include cell culture parameters such as pH, CO2, dissolved oxygen.  Sugar availability is driven by the media constituents and any feeds that are used.  Finally, lot-to-lot variation in commercially available supplements and feeds also introduce variation in glycosylation. It is important to select vendors and materials that have proven lot-to-lot consistency.

The wide variety of  factors impacting glycosylation can make controlling the quality profile a complex challenge.  Further, these same factors that drive post-translational modification also impact protein titer.  In many cases scientists will focus optimization efforts on either titer or glycosylation first, but find that both titer and glycosylation are affected by any one change.  In some cases, scientists will have to make a hard choice between quality and yield.  It is possible to focus on the physiology of the cell and target the ER and Golgi to increase both titer and consistency.


What impact does Cell-Ess have on glycosylation patterns? When added can your product affect fucosylation or galactosylation?

The Answer:

As mentioned, the approach with Cell-Ess is to target the physiological make-up of the ER and Golgi to effectively make them work more efficiently and function appropriately.  The hypothesis is if you are able to target the membrane constituents of the ER and Golgi, then there would be greater consistency and higher order glycoforms.  With the addition of Cell-Ess in two different media bases, we have seen increased consistency in the glycosylation pattern of monoclonal antibodies, suggesting that the Golgi is functioning more uniformly across groups to increase reproducibility.  Further, with the addition of Cell-Ess, we also observed increased galactosylation in two different base media, also suggesting more efficient Golgi.  The amount of glycoform G0F was decreased, masking the increase of fucosylation associated with higher order glycoforms, so the net result is a decrease in fucosylation.  In other work, we have shown a greater than 20% increase titer by increasing the amount of protein made per cell, which also points toward a more effective ER and Golgi.


In your description you mention that your products improves the consistency of glycosylation patterns. Have you seen this replicated and what kind of media are you adding your supplement to? I am just wondering about the consistency of the media and whether that would affect your results.

The Answer:

You ask an interesting question.  We did see the same effect of increased consistency in two different media bases.  Interestingly as you imply there were two different glycosylation patterns because the constituents in the base media were different but the increase in consistency of the glycoform patterns within each group remained the same.  We hypothesize that the effect would be similar throughout a variety of different media because the way Cell-Ess works is to improve the function of the Golgi and the ER which is one of the major drivers of glycan addition.  By improving the health of those organelles the consistency will improve.

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