Scientists tackle a key problem in drug manufacturing

Imagine making life-saving medicines more rapidly, less expensively and more efficiently. Biomanufacturing, which uses living cells to create medicines such as vaccines, antibodies and therapeutic proteins, is a cornerstone of modern healthcare. However, a major hurdle in this process is lactic acid – a waste product cells create as they produce energy. This build-up of lactic acid slows cell growth and reduces the amount of medicine made.

The cells most commonly used in biomanufacturing, called Chinese hamster ovary (CHO) cells, naturally produce lactic acid, reducing efficiency and increasing complexity. Now, researchers have developed a groundbreaking genetic technique to stop these cells from producing lactic acid. This innovation could produce drugs more rapidly, more easily and much more cost-effectively.

“This is a major step forward for biomanufacturing and beyond. By eliminating lactic acid production through genetic editing, we have given cells more freedom to work in various ways without affecting their growth or productivity. This does not just improve how we make medicines; it also lets us explore new scientific questions. We can now better understand how cells interact with their surroundings, whether in immune responses, cancer or other biological systems. The potential for new discoveries is huge,” explains first author Hooman Hefzi, Associate Professor, Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby.

Inefficient and wasteful

Mammalian cells, especially CHO cells, are widely used in biomanufacturing because they can produce complex, high-quality proteins for therapeutic use. However, a persistent challenge in biomanufacturing is managing cell growth and nutrient processing, especially the accumulation of lactic acid, a by-product of their metabolism.

“This stems from the way cells process energy, which is central to understanding this metabolic bottleneck. One key problem has been lactic acid – cells produce too much of it, and this has been a growth blocker for decades,” says Nathan Lewis, Georgia Research Alliance Eminent Scholar and Professor, University of Georgia, Athens, USA and Professor at the Department of Biotechnology and Biomedicine of the Technical University of Denmark.

Although this process supports rapid growth in some biological contexts, it poses significant challenges for manufacturing processes in which efficiency and consistency are critical.

“Lactic acid production is not unique to biomanufacturing; it reflects a broader metabolic strategy of rapidly growing cells across biological systems – including in embryos, immune responses and even tumours,” clarifies Nathan Lewis.

Instead of fully processing glucose into energy using oxygen, these cells convert it into lactic acid, even when oxygen is available. This approach enables rapid glucose consumption and supports growth, but the process is inefficient and produces excess waste.

“In the context of biomanufacturing, the accumulation of lactic acid has significant drawbacks. Improvements have largely come from adjusting how cells are grown, what they are fed or the equipment used. These changes reduce lactic acid production somewhat and increase protein production,” explains Nathan Lewis.

A delicate balancing act

To counteract this, buffering agents are added to the cell culture, but this increases osmolarity, further stressing the cells. This dual challenge reduces the amount of protein produced and can compromise the quality of the final therapeutic product. Managing and minimising lactic acid production has therefore been a key focus in optimising biomanufacturing processes.

“The production of lactic acid is closely tied to how cells set priorities for their energy pathways. When cells consume glucose, they produce pyruvate, which can either be processed efficiently in the mitochondria for maximum energy or converted into lactic acid through a less efficient pathway,” outlines Hooman Hefzi.

“The choice between these pathways is regulated by cellular energy levels and signalling molecules. In rapidly growing cells, the lactate pathway acts as a safety valve, enabling rapid glucose processing without overwhelming energy systems. You see it in yeast and bacteria, in which similar pathways often provide a growth advantage – they grow more rapidly, even if it is less efficient.”

This process, while beneficial in natural contexts such as rapid immune responses or embryonic development, creates challenges in controlled environments such as biomanufacturing. This creates a delicate balancing act for researchers and manufacturers.

“Over the decades, improvements in how cells are cultured – such as changes in nutrients, feeding strategies and environmental controls – have reduced lactic acid levels and enabled higher cell density and better protein yield. However, these strategies often address symptoms rather than the root cause of lactate production,” says Hooman Hefzi.

High risk

Many of the breakthroughs in biomanufacturing have come from tweaking external factors – such as how the cells are grown, what they are fed and how the bioreactors are set up. These changes have helped to reduce lactic acid production somewhat and enabled the cells to grow in larger numbers, producing more protein.

“Over the years, scientists noticed that as CHO cells grew more densely, they produced more protein. This trend followed a clear pattern, but it had started to plateau. That is when, at the Novo Nordisk Foundation Center for Biosustainability and National Biologics Facility Denmark, we decided to tackle cell metabolism as a key area for improvement,” highlights Nathan Lewis.

At that time, a student named Hooman Hefzi undertook a project to map all the energy and chemical pathways in CHO cells. During this work, he identified a potential bottleneck in lactic acid production. He proposed a bold idea: turning off five specific genes simultaneously could potentially stop the cells from producing lactic acid altogether.

“Hooman’s idea was high risk. Previous attempts to stop lactic acid production by targeting a key enzyme, lactate dehydrogenase, had failed because it killed the cells. But Hooman believed that targeting several parts of the system at once could disable the problem cleanly without harming the cells,” says Nathan Lewis.

Are the cells still alive?

“Rather than aiming for small improvement, such as a 30% reduction, we decided to go all-in and completely remove the feedback loop. This would give us a clear answer as to whether eliminating it entirely would solve the problem – and it did,” explains Hooman Hefzi.

Collaboration was established with Bjørn Voldborg, who was leading the CHO cell engineering team at the Novo Nordisk Foundation Center for Biosustainability. He carried out the complex task of knocking out all five genes at once, a step critical for testing the feasibility of the idea.

“CRISPR has revolutionised genetic engineering. Tasks that used to take years – such as turning off five genes at once – can now be done in months. Twenty years ago, people would have laughed at the idea, but now it is so much faster and simpler, enabling us to tackle bigger challenges more rapidly,” says Hooman Hefzi.

In this project, CRISPR technology was used like precise molecular scissors to cut out specific parts of the DNA. By targeting five genes at the same time, the process reprogrammed the cells to stop producing lactic acid.

“We were not sure whether the experiment would work, but just a couple of months later, Bjørn sent us the data: wild-type cells were making a lot of lactic acid as expected, but the modified cells were not making any at all – zero! We even joked, ‘Are the cells still alive?’ But he confirmed that they were alive and growing perfectly fine,” details Nathan Lewis.

“When Bjørn confirmed that this worked, we were amazed. We were concerned that removing lactic acid production might harm cell growth or protein production, but the modified cells grow just as well – or even better – since they no longer acidify their environment. The absence of lactic acid not only prevented harm but also improved cell performance in some cases. They produced as much protein as the original cells and, in some cases, even more,” says Hooman Hefzi.

A huge breakthrough

Eliminating lactic acid production appeared to indirectly enhance cell performance. To determine whether this approach could be applied to other types of cells, researchers tested it on a human cell line commonly used for manufacturing gene therapies and for basic research. The same genetic engineering strategy successfully stopped lactic acid production without harming the cells, demonstrating that the method could potentially be effective across types of cells.

“Controlling lactic acid production is a big challenge in biopharmaceutical manufacturing. Some companies handle it well, and others struggle. Eliminating lactic acid entirely enables us to bypass this issue, enabling more efficient processes and a focus on the next challenges in drug production,” states Hooman Hefzi.

“This research could save companies a lot of money. For instance, one company I worked with developed an expensive system – costing about one million dollars per reactor – to manage how cells produce lactic acid,” says Nathan Lewis.

By removing a few genes, researchers can achieve the same effect without relying on costly systems, making cell growth and production far more efficient.

“This is a huge breakthrough because we successfully turned off an essential gene – which should have killed the cells. Instead, we removed part of the cell’s internal system, eliminating lactic acid production entirely, which had never been achieved genetically before. This success not only opens new possibilities for biomanufacturing but also provides freedom to optimise processes in ways that were not possible before,” highlights Nathan Lewis.

Like playing whack-a-mole

The researchers are now studying what happens when cells stop producing lactic acid by examining how their internal processes change. For example, do the cells switch to new genetic programmes or redirect their metabolic pathways?

“Are they redirecting efforts to different metabolic pathways? We noticed that some key molecules involved in metabolism increase when lactic acid production stops, which could have far-reaching effects. This is a bit like playing whack-a-mole – when you solve one problem, another one pops up. But overall, we have seen a big improvement in how the cells perform,” explains Hooman Hefzi.

Scientists have debated why cells rely on lactic acid production. Some argue that this is because it helps cells to produce energy quickly, like a short burst of power. Others think that this is because this process helps to make the building blocks cells need to grow.

“This work does not just improve how we grow cells for biomanufacturing – it helps us to understand how cells interact in such areas as developmental biology, immune responses and tissue function. Exploring these communication pathways could lead to breakthroughs in many areas,” states Nathan Lewis.

“By stopping lactic acid production, we have given the cells more flexibility to explore different states. The cells naturally find a state that works for them, but this opens new possibilities for improving their performance and understanding their biology,” explains Hooman Hefzi.

Making drugs more rapidly and more efficiently

Cells naturally communicate with each other as they grow, sensing their environment and adjusting how they act. By targeting signals such as lactic acid, which limits growth, researchers can fundamentally alter how cells communicate and respond to their surroundings.

“Now we can ask much deeper questions about how cells act in various situations, such as in embryos, tumours or immune systems. For example, we can study how cells interact with their environment or make it more acidic, which could affect how they move. This deeper understanding could provide new answers about how cells work and interact,” says Nathan Lewis.

The new findings open exciting opportunities. By removing key signalling molecules from cells, researchers can determine how these changes affect how cells interact and communicate with each other and their surroundings.

“So we think – well, we hope – that this turns out to be a generalisable strategy to eliminate the Warburg effect. With this approach, we can really start to directly explore some fundamental biological questions. What is the actual purpose of the Warburg effect? How does it affect the state of the cell and its physiology? And now, we have a clean comparison – a cell line that grows just as well but does not produce lactate,” interprets Hooman Hefzi.

The current hunch is that eliminating lactic acid production creates a metabolic ripple effect. It redirects the cell’s efforts, leading to increased production of key molecules such as acetyl coenzyme A. These changes can affect gene regulation and protein behaviour, potentially providing new insight into how cells function and adapt.

“Removing lactic acid production does not make cells grow forever – it just removes the first barrier in the process. This enables more intense production methods, manufacturing drugs more rapidly and more efficiently, although it also introduces new challenges that need to be overcome,” concludes Hooman Hefzi.

“Multiplex genome editing eliminates lactate production without impacting growth rate in mammalian cells” has been published in Nature Metabolism. This work was supported by the United States National Institute of General Medical Sciences, the European Union’s Horizon 2020 research and innovation programme, the Austrian Research Promotion Agency, the Austrian Science Fund, doc.funds (Austrian Science Fund), a Christian Doppler Laboratory grant for recombinant protein production in mammalian cells and the Novo Nordisk Foundation through the Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark.