Post-translational modification is a critical step in the synthesis of biotherapeutic molecules. Among these alterations to the original naked protein, glycosylation may be the most significant and difficult to properly complete. In this roundtable, we discuss the challenges and the pitfalls involved in generating authentic glycosylated molecules in cell culture and microbial production facilities. GEN interviewed several authorities in the field of protein production, including:

Steve King, president, Artius Bioconsulting
Sylvain Marcel, PhD, vice president, protein expression sciences, iBio
Andrew Bulpin, PhD, head of process solutions at MilliporeSigma
Vince Nguyen, senior upstream process development engineer, Precision Biosciences
Catriona Thomson, PhD, director, R&D and technical services, Sartorius Stedim Biotech


GEN: Accurate glycosylation is essential for proper functioning of monoclonal antibodies (mAbs) generated in vitro. What are the advantages and disadvantages of using engineered bacterial or yeast cell lines to generate authentic mAbs that can function in a clinical setting as opposed to an engineered mammalian cell line (such as CHO)?

SK: Engineered bacteria or yeast cell lines can have a number of potential benefits, including the consistency of glycosylation, speed of development associated with production clone selection, higher density growth, and potentially a better cost of goods. In particular, the ability to tightly control glycosylation can allow a drug developer to finely tune effector functions which could potentially lead to more consistent clinical performance.

CT: Advantages include lower production costs, and an easier way to manipulate the genome. Furthermore, the investigator can more predictably manipulate the glycosylation profile based on the types of enzymes introduced into the bacterial or yeast strain. While these alterations can be achieved in mammalian cell lines, yeast and bacteria are superior to mammalian cells in many situations due to their smaller genomes, the wide choice of engineered strains, and a long history of modification, On the negative side, among the disadvantages are the fact that upfront manipulation and engineering will invariably be required, bringing potential stability issues into play. Finally, there will be the necessity to demonstrate that only the desired traits have been introduced and that nothing unwanted has crept in.

SM: Engineered glycosylation systems are an emerging development with the potential to allow companies a new option for expressing antibodies with desired glycosylation and associated potency properties. We are working with a plant expression system using N. benthamiana which has been evolved to include glycan engineering. In fact, some transgenic plants now possess human glycosylation pathways, e.g., the human beta-1,4 galactose capping of the glycan core to form G1 and G2 glycoforms or the human sialylation pathway. Other transgenic plants have been designed to avoid core fucosylation, an attractive method to produce afucosylated anticancer antibodies with enhanced ADCC activity.


GEN: Another alternative for producing glycosylated antibodies is enzymatically binding the naked antibody to the appropriate oligosaccharide molecule. How does this approach stack up against the biological synthesis of a glycosylated antibody?

SK: These systems can also allow the drug developer to achieve consistent glycosylation and antibody effector functions. The main drawback is that this approach requires additional processing steps which can result in lower overall process yield and the need to demonstrate appropriate clearance of the enzymes. Of course, if the developer already has an antibody with desired attributes then this approach could be the most efficient way to advance the molecule without the need to redevelop the expression system which could be a substantial advantage.

CT: Chemical synthesis is more controllable in terms of the type of glycosylation that is added, but there is the potential for improper conjugation of the oligosaccharide to the mAb. This may require additional testing to monitor the amount of correctly and fully glycosylated antibody in a similar way to calculating and controlling the drug-antibody ratio (DAR) of an antibody-drug conjugate (ADC).

In addition, chemical synthesis imposes a requirement for more steps for manufacturing: upstream mAb manufacture by cell culture, followed by mAb purification 1; mAb modification; and a second mAb purification step. In a cost/benefit analysis, the investigator may find that the gain in functionality of the fully glycosylated mAb might not exceed the loss in amount of material incurred during production and the increased resources required.

SM: Use of enzymes to alter glycosylation requires a further step in the process, with attendant disadvantages in terms of process complexity, potential yield losses, longer timelines, and extra steps in product validation. However, significant advancements were made recently to provide customization of N-glycosylation in vitro leading to homogeneous glycoforms. Although this approach is still considered too expensive to implement at scale, it is believed that the implementation of such methods will become more affordable and available as oligosaccharide donors are produced at larger scale with simpler methods (Noguchi M., Tanaka T., Gyakushi H., Kobayashi A., Shoda S. J. Org. Chem. 2009;74:2210–2212. doi: 10.1021/jo8024708). This procedure, combined with an in vivo approach to produce the acceptor proteins, such as deglycosylated mAbs using endoglycosidase H co-expressed in plants, may represent opportunities for large-scale manufacturing (Bennett et al. Int. J. Mol. Sci. 2018, 19(2), 421 10.3390/ijms19020421).


GEN: According to Ultee and Easton (BioPharm International (28 {11}: 20–25, 2015), “The choice of cell clone affects product quality. Each clone has slightly different abilities for glycosylation, and viabilities vary. Therefore, it may be necessary to select a cell clone that is not the highest producer to achieve the desired protein quality.” How do you view this trade off and what are your strategies for managing it in an optimal fashion?

AB: Protein quality is an important consideration in clone selection and, in some instances, may be prioritized at the expense of other clone characteristics. However, rather than mitigate the trade off, it may be more prudent to employ methods throughout cell line and process development to attain both protein quality and production goals.

Firstly, high throughput screening of critical quality attributes, both at early and late stages in cell line development, improves the identification of ideal clones. Incorporating protein quality screening on stable pools and cloning from pools that most closely match desired critical quality attributes increases the likelihood of identifying clones that match key criteria. Additionally, the ability to incorporate protein quality screens into preliminary clone assays better ensures that the final clone fits the preferred protein characteristics.

Secondly, performing process development on multiple top clones may help ensure that both titer and protein quality goals are met. While each clone may be predisposed toward certain profiles in terms of charge heterogeneity, glycosylation, and aggregation of the recombinant biotherapeutic, it is important to keep in mind that these critical quality attributes may be impacted by process parameters. The ability to screen more than one candidate clone in a variety of process conditions may help balance these two aspects of clone performance: titer and protein quality.

Finally, the higher producing clones that are generated in a cell line development process, the better the chances are that a clone can be isolated that matches both the favorable critical quality attributes and the desired productivity levels. A robust cell line development process that generates multiple high expressing clones eliminates the need to sacrifice titer for critical quality attributes.

SK: The identification of critical quality attributes early on is important in drug development, especially if they relate to effector functions. Having a defined product profile that can guide clone selection is ideal although this aspect of clone selection is often overlooked in early drug development. The key is to identify the desired product quality attributes prior to clone selection and subsequent process media optimization so that you can achieve the optimal process for both yield and product characteristics.

CT: Understanding this tradeoff is a pragmatic way to approach one of the many facets of clone selection. Factors to consider include the type of molecule that is to be produced and what the CQAs are in relation to the glycoprofile. For example, in a biosimilar molecule, there is a known glycoprofile at which to aim and it is prudent to consider sacrificing production quantity for protein quality. This may mean selection of a clone that more closely matches the innovator glycosylation profile at the outset and working on media optimization to increase productivity during process development. With an NBE there is more “flexibility” in terms of not having to match a pre-existing glycan profile, but particular glycans present on certain clones might be less desirable than others. For example, clones with high levels of increased mannose structures may be more likely to be cleared from the body faster, and particular types of sialyation increased risk of unwanted immunogenicity.

SM: Alternative expression systems such as a transient expression applied at large scale may be an appropriate response to this issue. The plant transient expression system, applied at small and large scale, does not require any cell-line development or scale-up studies. Because the bioreactor unit is a single plant, the batch size corresponds to the number of plants being transfected at scale. The product quality is thus not by design affected by the clone or batch size because the dynamics of expression does not change as it remains a single plant.

VN: Much of the time, the ability to select clones based on glycosylation is derived from the reality of development timelines and the particular needs of the program. I would suggest including not only titer but also effector function indicating product characteristics such as fucosylation. Other process parameters can be manipulated later during development to control glycosylation to reach desired effector function but clearly selecting clones for optimal product characteristics from the beginning makes sense. (Note: Dr. Nguyen wishes to point out that the opinions he expresses are his own, and do not reflect the policies of Precision Biosciences).


GEN: According to Reinhart, et al. (Appl Microbiol Biotechnol. 2015. 99(11):4645-57), the choice of cell culture medium can profoundly affect the quality and quantity of antibody glycosylation, possibly linked to amino acid levels and other properties of the medium in question. Could you discuss your experiences with different media choices as to their effects on glycosylation of monoclonal antibodies?

AB: Media selection, cell line selection, genetic engineering, and process optimization are all levers used to target specific glycosylation profiles in recombinant biotherapeutic molecules. The composition of the medium can impact the glycoprofile. It is not so simple as ensuring high quantities of the required substrates (for example, adding galactose to increase galactosylation.) Multiple components can impact each step along the glycosylation pathway, and there may be complex interactions at play that influence the effects of those components.

One approach that MilliporeSigma takes to achieve a specific glycoprofile is through custom media development, in which the impact of each media component is interrogated using high throughput experiments and advanced statistical tools. In the case that the composition of the media is unknown, or in the case that a large scale optimization experiment is not possible, additives such as the EX-CELL® Glycosylation Adjust (Gal+) can be applied to hit the targeted profile.

Another aspect to keep in mind is the role of raw materials. Lot-to-lot variation in raw materials can impact the glycoprofile and ensuring that the media supplier has a robust raw material characterization program in place to identify these variations can help ensure that glycoprofiles remain at the target throughout scale up and over time.


CT: The composition of the medium is incredibly important in the final glycoprofile of a therapeutic. Modification of the observed glycan profile can be achieved by supplementing or modifying media. This complex process can be expedited by use of DOE and the Sartorius Ambr technology to more quickly reach the desired profile.

The molecules produced each have their own pattern of glycosylation. This pattern can be varied to a certain extent by the choice of cell line to express the appropriate molecule. We have found that mAbs could be identified and grouped by their glycoprofile independent of the type of CHO cell line that was applied. Differences from cell line to cell line were detectable, but the general pattern stayed the same, if we kept the media system constant. When we changed the media system for mAb expression, the mAb pattern changed significantly in the same direction, independent of the CHO strain that was applied for production.

Thus the impact of the media and the manufacturing process was much more significant than the cellular background.


GEN: Chang, et al. (Nat Chem Biol. 2019;15(7):730-736) states: “Precise and rational modification of N-glycosylation will allow new recombinant protein therapeutics with tailored in vitro and in vivo effects for various biotechnological and biomedical applications.” They have investigated the role of serum level modification in the study of serum free media. Do you have experience in this area, and could you comment on your observations?

SM: I have experience with modern technologies for mAb production in plants engineered for appropriate glycosylation of mAbs which does not involve serum or animal-derived media.

VN: There are trace metals that increase fucosylation level because they are inhibitors of cofactors for enzymes that are responsible for fucose. It is a good practice to monitor levels of these trace metals in water and other raw materials in the effort to control critical product characteristics such as fucosylation.

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