Some years ago, I came across a study that stopped me in my tracks. Cyclists who ate pistachios before a 75-kilometer time trial performed worse than those who didn't. Not marginally—4.8% slower. Statistically significant. Why would a healthy nut sabotage athletic performance?

The culprit: raffinose, a trisaccharide in pistachios. During intense exercise, your gut becomes more permeable—and raffinose from the colon leaked into the bloodstream. Once there, it correlated with oxidative stress and leukotoxic effects. Your body was literally fighting the fuel you just gave it.

This concept is discussed in greater detail in the Smart Biotech Scientist Podcast, hosted by David Brühlmann, founder of Brühlmann Consulting.

I thought: if raffinose can slip through a stressed gut barrier and trigger metabolic effects in humans, what does it do to CHO cells under bioprocess stress? That question led to one of the most surprising findings of my PhD work.

Before we dive into that story, let me give you the roadmap for today. First, I'll walk you through why glycosylation matters for biosimilars and what happens when it goes wrong. Then, I'll show you the biology of how raffinose changes glycan processing. After that, I'll take you through the experimental journey—what we tried, what failed, and what finally worked. And I'll close with the real-world impact this had on actual development programs.

But let me tell you what this episode is really about. It's about media design that increased high mannose glycans 2.8-fold in CHO cell culture runs. It works in multiple cell lines. It doesn't require cell line reengineering or expensive glycosidase inhibitors. It's not a universal fix—I'll tell you exactly when it works and when it doesn't—but if you're 6 to 12 months from filing, this could save your timeline.

Glycosylation 101

Before we go further, a quick primer for anyone who's new to glycosylation or needs a refresher.

Glycosylation is the process where sugar chains get attached to your protein as it's being made. Think of it like decorating a cake as it comes out of the oven—except the decorations determine whether your drug works or gets cleared from the bloodstream in 10 minutes.

This happens in two cellular compartments. First, the endoplasmic reticulum adds a core oligosaccharide structure—14 sugars arranged in a specific tree-like pattern. That's the starting template. Then, the protein moves to the Golgi apparatus, where the real action happens. Enzymes in the Golgi trim off some sugars and add others, sculpting that core structure into the final glycan pattern.

For monoclonal antibodies, glycosylation affects a lot. Half-life in circulation. Immune activation through Fc receptors. Protein stability. And most importantly for biosimilar developers: whether regulators will approve your product.

Get it wrong, and you're looking at 6 to 12 months of rework. Get it right, and you're on track for approval.

Now, back to raffinose and why it matters.

The Glycosylation Problem

Let me paint you a scenario that's all too common in biosimilar development.

You've spent 18 months developing a biosimilar mAb. Your cell line is stable. Titer is great—3 grams per liter. You're hitting your productivity targets. The team is feeling good.

Then analytical drops the bomb. Your high mannose levels are 1.4 percent. The reference product? 6 percent. You're out of spec.

Now, you might be thinking: why does this matter? It's just a small difference in sugar composition.

Here's why it matters. High mannose glycans affect antibody-dependent cellular cytotoxicity—ADCC. They affect receptor binding kinetics. They affect serum clearance rates. And most critically, they affect regulatory comparability. If your glycan profile doesn't match the reference product within an acceptable range, regulators will ask for additional studies. Or they'll reject your filing outright.

So what are your options?

1️⃣ Option one is a temperature downshift to ~32–33 °C. It can push the glycan profile toward higher high mannose, but the productivity tradeoff is clone- and process-dependent.

2️⃣Option two: kifunensine. It's a glycosidase inhibitor that blocks mannosidase enzymes in the Golgi. It's effective—you get high mannose, no question. But it's expensive, hard to scale, and regulatory agencies scrutinize any process that uses enzyme inhibitors. You'll spend months justifying it in your CMC package.

3️⃣Option three: re-engineer your cell line. Go back to the drawing board. Select a new clone with better glycosylation characteristics. This works, but it adds 12 to 18 months to your timeline. If you're racing to market against competitor biosimilars, that delay could cost you hundreds of millions in lost revenue.

What you really need is a media-based solution. Something that scales. Something that doesn't torpedo your productivity. Something that regulators understand because it's just a media component adjustment.

That's where raffinose comes in.

How Raffinose Works

Let me explain the biology of what's happening when you add raffinose to your cell culture.

Raffinose is a trisaccharide—three sugars linked together: galactose, glucose, and fructose. CHO cells can't metabolize it efficiently, so it accumulates in the culture medium and in the cell.

It turns out that raffinose competes with a specific enzyme in the Golgi: N-acetylglucosaminyltransferase, or GlcNAc transferase. This enzyme is responsible for adding the first branch to the core glycan structure. Think of it as the fork in the road where a simple glycan becomes a complex, multi-antennary structure.

When raffinose is present, it binds to the active site of GlcNAc transferase. It doesn't shut the enzyme down completely—it just slows it down. The result? Your antibody ends up with Man5—mannose-5—instead of the complex, fully elaborated glycans you'd normally see.

This is different from kifunensine. Kifunensine blocks mannosidase I and gives you Man8 and Man9—much earlier intermediates. Raffinose arrests glycan processing at a later step, giving you predominantly Man5.

We confirmed this mechanism with transcriptomics. When we looked at gene expression in cells treated with raffinose, we saw downregulation of galactosyltransferase—GalT—and other late-stage Golgi enzymes. The cells were adapting to the metabolic stress by dialing back their glycan elaboration machinery.

The key thing to understand is this: raffinose doesn't shut down glycosylation entirely. It arrests it at a specific step. That's why you get Man5 enrichment, not a complete block. And that's why it's tunable—you can dial the concentration up or down to hit your target glycan profile.

Now, knowing the mechanism is one thing. Making it work in practice? That's where things got interesting.

The Experimental Journey

Let me take you through what we actually tried in the lab. Because the path to the breakthrough was not straightforward.

1️⃣ Attempt one: standard supplements.

We started with the usual suspects. Manganese chloride—known to affect glycosyltransferase activity. Galactose and GlcNAc at various concentrations—trying to push or pull the glycan pathway. We tested these in 96-well plates across a range of concentrations.

Result? Minor glycan shifts. Nothing reproducible. Batch-to-batch variability was all over the place. No clear dose-response relationship. We were tweaking the system, but we weren't controlling it.

2️⃣ Attempt two: osmolality manipulation.

We knew that osmotic stress affects Golgi function. So we tried increasing osmolality by adding raffinose at high concentrations—50, 75, 100 millimolar. The idea was to stress the Golgi and slow down glycan processing.

Result? Growth crashed. At 50 millimolar raffinose, cell viability dropped below 70 percent by day 5. Titer tanked. We thought we'd hit a dead end. Raffinose looked like a non-starter.

But then we had a realization. We were confounding two variables: raffinose concentration and osmotic stress. High raffinose meant high osmolality, and we couldn't tell which factor was causing the growth inhibition.

3️⃣ Attempt three: the constant osmolality pivot.

This was the key experiment. We adjusted sodium chloride in the medium to keep osmolality constant at 315 milliosmoles per kilogram—regardless of raffinose concentration. So when we added 30 millimolar raffinose, we subtracted enough NaCl to maintain constant osmolality.

Result? Growth recovered. Viability stayed above 90 percent through day 14. And now we could test raffinose concentrations up to 100 millimolar without killing the cells.

Once we isolated raffinose's effect from osmotic stress, the data became crystal clear. We had a dose-response curve. We had reproducibility. We had scale-up potential.

It turns out that the breakthrough wasn't in finding a magic ingredient. It was in controlling the variables so we could see what raffinose was actually doing.

The Breakthrough

Here's what worked.

50 millimolar raffinose increased high mannose 2.8-fold compared to the control. The effect was robust. It worked in two different cell lines. It worked in two different basal media. It worked at every scale we tested: 96-well plates, shake tubes, benchtop bioreactors.

The surprise? The glycan profile was predominantly Man5. Not Man8 or Man9 like you'd see with kifunensine. This told us we were hitting a different step in the Golgi processing pathway.

Now, I was fortunate to apply this approach in actual development projects. I can't share specifics—those programs are confidential—but I can tell you this: when you're staring at a glycan mismatch 8 months before your IND filing, having a validated media lever in your back pocket is the difference between making your timeline and explaining to leadership why you need another year.

One more thing about this approach that's often overlooked: it's regulatory-friendly. Raffinose is a simple trisaccharide. It's available as a GMP-grade material from multiple suppliers. It's not an enzyme inhibitor. It's not a genetic modification. It's a media optimization—something process development teams do all the time.

If you're pre-IND, this is straightforward. You're optimizing your process, and media composition is part of that. Document it. Include it in your development report. Done.

Closing

So we proved raffinose works. But here's the hard part: how do you actually implement this in your process without spending 6 months on design-of-experiments studies?

In Part 2, I'll give you the exact decision tree. When to use raffinose. When it won't work. And the three experiments you need to de-risk it before committing to your manufacturing campaign.

If you want to dig into the full methods and data, the paper was published in the Journal of Biotechnology, 2017, volume 252, pages 32 to 42. DOI: 10.1016/j.jbiotec.2017.04.026.

Thanks for joining me in exploring the biology behind raffinose and glycan control in CHO cell culture.

If you found this episode valuable, I'd love your feedback. The best way to share it is by leaving a review. It helps other scientists discover these insights and lets me know what's resonating with you.

See you in Part 2.

Your Next Step

Book a free consultation to help you get started on any questions you may have about bioprocess development: https://bruehlmann-consulting.com/call

David Brühlmann is a strategic advisor who helps C-level biotech leaders reduce development and manufacturing costs to make life-saving therapies accessible to more patients worldwide.

He is also a biotech technology innovation coach, technology transfer leader, and host of the Smart Biotech Scientist podcast—the go-to podcast for biotech scientists who want to master biopharma CMC development and biomanufacturing.  

Hear It From The Horse’s Mouth

Want to listen to the full interview? Go to Smart Biotech Scientist Podcast. 

Want to hear more? Do visit the podcast page and check out other episodes. 
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