Hi Reader, last week I posted about an essential step to make your lab greener: Quantification.
I thought this post might be of interest to you - also because I share some of my experience in the comments.
And while we often overlook the benefits of apparently trivial actions like quantification, the same can be said about innovations.
A good example is trypsin alternatives — let me explain the issue:
Today's Lesson: Trypsin Alternatives
How enzymes can be made more sustainable.
Number Of The Day
According to several sources such as Market Research Intellect or Valuates Reports, the animal-free trypsin market is valued at $80–150 million in 2024. That’s quite a sum and potentially bigger than numbers on traditional animal-based trypsin. With an annual growth rate of 5–8.5%, this topic is becoming increasingly relevant. What’s driving this growth, and will it actually benefit the environment? Let’s take a closer look.
80 Million
Exploring Trypsin Alternatives
We all await more green innovations. However, they’re sometimes already available.
We just have not heard about them... But why?
On the very left, you see Corning’s new culture flask, which reduces plastic use by more than 20% — something I only discovered while actively researching them. The trypsin alternative shown is a mock-up from Novonesis, and the serum-free medium was covered in one of my previous lessons.
Two reasons:
Poor visibility – Not every sustainable product is sufficiently advertised.
Hidden benefits – Sometimes the advantage is there, but not obvious at first glance.
Trypsin alternatives might suffer from both but while we can do little about A), let's find out how to understand and overcome B).
Why, How, and When Trypsin?
Trypsin is best known in cell culture for detaching adherent cells. But it’s also used to digest proteins and peptides for MS analysis, or in recombinant insulin manufacturing to convert proinsulin into active insulin.
In the industry, R&D labs might use similar techniques, whereas large-scale production obviously relies on reactors—or at least high-volume, high-surface flasks—to significantly increase throughput. If you like to know more, here is a YouTube video on splitting cells.
Since cell detachment is critical in processes like vaccine production — and due to its role in insulin manufacturing, we see robust demand from the growing pharmaceutical industry.
However, behind the scenes, it comes with a few concerns:
It's traditionally extracted from the pancreas of pigs or cows, requiring their slaughter.
Quality can vary since it comes from living animals.
It carries risks of contamination (viruses, prions), which is especially critical in stem cell or pharmaceutical applications.
In short: effective, yes — but it comes with ethical and scientific challenges.
The Innovation
Recombinant trypsin, produced without animals using engineered microbes, could be the solution.
Whether Cellseco, Sartorius or biosera, there are dozens of vendors you can purchase recombinant Trypsin from.
Most commonly, these alternatives are made by fermentation in Pichia pastoris (a type of yeast) grown in bioreactors or from bacteria such as E.coli. Trypsin is then isolated, purified, and stabilized.
For example, TrypLE™ functions just like trypsin. However, it is stable at room temperature (up to 24 months), gentler, and doesn’t require inhibitors for inactivation (dilution suffices).
This data stems from a white paper by Nestler and colleagues created already in 2004. TrypLE™ Express shows that even after 8 weeks at 37°C, 50% enzyme activity remains – samples were stored in the dark. Of note, I am not sure why some bars do not show error bars while in others they are apparently not middle-aligned... Anyway, rPU stands for Recombinant protease activity unit, denoting enzyme activity.
Other alternatives, like r-Ac-trypsin (acetylated recombinant trypsin), are optimized for proteomics: they offer higher activity, more stability, and are less prone to autolysis.
The Sustainability Angle
These products offer several clear advantages:
No animal use → relies on microbes instead of slaughtered animals
No contamination risk → fewer failed batches and less repetition
More consistent activity and less autolysis → better reproducibility
Room-temperature stability → reduces energy demand for cooling
Inactivation by dilution → no need for added inhibitors
All of this points to less waste and potentially fewer resources used downstream.
Manira et al. studied how Trypsin-EDTA (TE) and recombinant trypsin, TrypLE Select (TS), perform across two cell passages. They report:“The results of our study showed that the total cell yield for both keratinocytes and fibroblasts treated with TE or TS were comparable… In conclusion, the performance of the recombinant trypsin is comparable with the well-established animal-derived trypsin for human skin cell culture expansion in terms of cell yield and expression of specific cellular markers.”The image shows antibody-stained cells (n=6), manually counted, with characteristic protein markers for each cell type.
The Sustainability Challenge
However, let's not jump to conclusions. A few sustainability considerations remain:
Recombinant production also consumes energy and chemicals — fermenters, purification steps, sterilization. It’s not footprint-free.
Animal-based trypsin often comes from meat industry by-products. Using it doesn’t necessarily increase animal harm.
Its production sites may be centralized (e.g., in the US), leading to longer transportation routes and associated emissions.
Applying The Knowledge
So, is recombinant trypsin truly more sustainable?
We don't know — because there are no LCAs (life cycle assessments) or carbon footprint analyses available.
But there may be sustainability advantages beyond the environmental:
Scientifically more sustainable: better reproducibility, less contamination, and less auto-degradation
Operationally more sustainable: easier to store and use, saving time and energy
For non-critical tasks like routine cell detachment, switching should be low risk.
Tip: Click to enlarge. This is another graph from the white paper by Nestler and colleagues, showing an HPLC chromatograph – indicating several impurities on the porcine Trypsin due to several side peaks.
For more sensitive processes, switching might require validation, but could improve efficiency or standardization in digestion workflows – aligning better with GMP standards.
Key Takeaway: We need to investigate sustainability claims — not all are accurate but sometimes, the real benefits are hiding where marketers don’t even look.
Upcoming Lesson:
Carbon Footprint of Imprecise Science
How We Feel Today
References
Manira, M. et al., Comparison of the effects between animal-derived trypsin and recombinant trypsin on human skin cells proliferation, gene and protein expression. Cell and Tissue Banking, 2014, 15(1), 41–49. doi:10.1007/s10561-013-9368-y
Wu, F. et al., Recombinant acetylated trypsin demonstrates superior stability and higher activity than commercial products in quantitative proteomics studies. Rapid Communications in Mass Spectrometry, 2016. doi:10.1002/rcm.7535
Karbalaei, M. et al., Pichia pastoris: A highly successful expression system for optimal synthesis of heterologous proteins. Journal of Cellular Physiology, 2020. doi:10.1002/jcp.29583
Li, C. et al., Development of an effective method for purifying trypsin using a recombinant inhibitor. Protein Expression and Purification, 2025, 225, 106597. doi:10.1016/j.pep.2024.106597
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