When working on protein purification, I’ve noticed something interesting: Scaling from a small test prep to bulk quantities isn’t just “more of the same.” It’s a whole new game with unexpected roadblocks. i) Expression yield might suddenly drop. ii) Columns clog. iii) Protein stability becomes unpredictable. iv) Even storage turns into a science experiment. I’m kicking off a series on protein purification challenges as a conversation in ResearchGate and also in LinkedIn. I would like to know more about real-world hurdles that my peers have been facing and brainstorm solutions together with them, other experienced researchers and those just stepping into this space. Whether it’s about equipment limitations, buffer prep headaches, stability issues, or something else entirely. Please share it in the comments. Let’s see what patterns emerge, and maybe we can solve a few of them along the way.
LinkedIn link of the same discussion : https://www.linkedin.com/feed/update/urn:li:activity:7360018718539571201/
Dear Seema,
In general if you purify the protein on large scale , you faced lots of challenges. In your discussion you are not says the source from which you aimed to purify the protein.
Protein stability, impurities removing, Protein Aggregation preventing, Process optimization, limitation to chosen chromatography techniques, Recovery rates of protein and very important is Post translation modification of proteins (active confirmation of proteins that is purified protein maintains its activity)
Article Challenges and solutions for the downstream purification of ...
Please refer this article, you got the sufficient information.
Tole S.B. Thanks a lot for sharing the article.
You’re spot on - scaling protein purification isn’t just “bigger volumes,” it’s a whole new problem set. The source system matters a lot (bacterial vs. insect vs. mammalian), and issues like aggregation, activity loss, and impurities become more pronounced.
Some key hurdles I’ve seen:
1. Expression levels shift at scale due to oxygen/nutrient limits.
2. Columns clog or foul faster with large volumes.
3. Buffers are harder to standardize consistently.
4. Preserving active conformation is harder than just chasing yield.
Thanks for pointing to the downstream purification article, that’s a solid reference. I’d love to hear: what was the most unexpected challenge you faced when moving from test prep to scale?
Seema Nath, The most unexpected challenges when scaling up protein purification are the discrepancies between small-scale and large-scale processes, such as sudden drops in protein yield, increased protein aggregation, protein instability over time, unexpected column clogging, and significant difficulties with buffer preparation and equipment limitations.
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