Modification of Glycans in Bioprocessing
with Ryan Boniface
Protein quality determines clinical behavior. Glycosylation is a key product quality attribute for many biotherapeutic proteins expressed by mammalian cells. N-linked glycans can display macro- and microheterogeneity with a degree of variation that depends on several factors. It’s often challenging to predict, achieve, and maintain preferred glycosylation profiles. For an “Ask the Expert” webinar on 15 June 2016, bioproduction scientist Ryan Boniface of Thermo Fisher Scientific explored approaches to modifying glycan patterns.
Boniface’s Presentation
Thermo Fisher is strongly focused on enhancing protein quality both through cell culture process and media. The glycosylation profile of a protein is an important parameter that affects its structural and functional characteristics as well as in vivo half-life, efficacy, metabolic activity, and clearance rates.
Cell growth and product titer don’t matter if expressed product is low in quality. Widely published factors that affect glycosylation profiles generally fall into two categories: process parameters and media and feed components. Examples include cell harvest viability and changes in glutamine concentration that alter ammonia levels. Both can affect glycan profiles.
We studied the effects of glycosylation-modifying components added as a direct supplement titrated into a culture. Such additions changed glycan profiles significantly. However, they led to unpredictable shifts in glycosylation from G0F to G1F and G2F. The unpredictable results do not indicate at what point supplementation should be added to a culture, which could necessitate multiple rounds of titration to find specific levels. So we dropped the additive supplemental approach in favor of developing a more complete feed supplement incorporating those glycosylation modulating components with a new feeding strategy.
Our new strategy involved “transitioning” from an EfficientFeed+ product to GlycanTune feed during a culture. For controls, we fed only EfficientFeed+ to one batch and only GlycanTune feed to another. The former would mostly give a G0 peak; the latter would ensure maximum galactosylation and shift glycoforms from G0 to G1F and G2F. Our transition points would fall between those extremes.
Feeding began with EfficientFeed+ media, with the GlycanTune switch timed to induce predictable changes in glycan profiles. A transition early in culture (e.g., day five) would increase galactosylation (G1F and G2F), whereas a later transition would keep a largely G0F peak. Protein quality improved (decreasing G0F) by almost 45% and shifted primary glycans to G1F and G2F. At each transition point (depending on the day), we got a linear, stepwise progression that made it easy for us to target glycan profiles. With a predictable linear response, unnecessary process development steps can be minimized.
What makes this a powerful tool is that by starting with EfficientFeed+ media and transitioning to a GlycanTune product, we could match the glycan profiles of four commercially relevant biotherapeutic molecules. That shows predictable and precise targeting of glycan profiles. We gained a linear glycan response and improved protein quality by reducing G0F by ≤45% and shifting the primary glycans to G1F and G2F. This modulation has no negative influence on cell growth or titer.
Questions and Answers
Does this feed affect other product-quality attributes? We are now looking at some others, including charge variance and protein aggregation.
Does the new product affect productivity? None of the three GlycanTune feeds (GlycanTune A+, B+, and C+) negatively affects titer.
Have you tested GlycanTune with other media? Not yet, but it is something we can test. We perform applications with many of our feeds to see how they perform with other media, both internally and externally.
Why does ammonia concentration affect glycosylation? Increased ammonia concentration provides generally unfavorable conditions for cell cultures, putting stress on the cells. This often happens at the end of a culture. The decrease in terminal glycosylation mostly comes from the inhibitory affects and toxicity of ammonia.
How do sialylation and galactosylation compare? In my examples, our cell line does not sialylate. However, better terminal galactosylation should create a better base and more sites available for terminal sialylation. This is something that we will be looking into.
What analytical methods have you used to determine the galactosylation profile? We have an internal method that uses an aminopyrene trisulfonic acid (APTS) dye with glycan digestion. We can run a test on our ABI 3500 Genetic Analyzer system using our new “GlycanAssure” APTS method. It reduces the time to get glycan peaks from 24–48 hours down to seven to nine hours.
What is the recommended strategy to increase G0? Our current EfficientFeed products don’t modulate glycans. By using them, a customer could start with a high G0F peak baseline and very little terminal galactosylation. We currently haven’t worked on any inhibitory products
More Online
The full presentation of this “Ask the Expert” webcast can be found on the BioProcess International website at www.bioprocessintl.com/webinars/modification-of-glycans-in-bioprocessing. And you can visit www.thermofisher.com/glycantune for more information.