Hello!
We are producing a recombinant protein (from mammal cells) and we need a 6xHis tag for the purification. This protein is aim to be used in preclinical experiments in the short-medium term, but I can't find anywhere if the 6xHis tag is compatible with the FDA guidelines or we have to cut it out.
Some considerations I came across:
- Histidine is actually a very common aminoacid.
- The insertion of TEV cleaving site will anyway leave some extra aa on our protein and, on top of it, it will add an extra step in the manufacturing that has to be validated.
Does anyone knows where to find the official FDA guideline for this topic or the list of tolerated tags?
Many thanks!
Fil
As far as I'm aware, there is no explicit rule against that. In practice people don't do that for process reasons.
His tags are great for getting many different proteins in one system, quick & dirty, usually from E. coli. In mammalian cells I would not usually choose it even in the research setting. Too many metal binding proteins co-purify.
Once you get into thinking at a therapeutic scale -- what is really the safest, cleanest, most scalable, and cost effective way of making your specific protein, I can't imagine your final selection would include a his-tag. It will certainly be a multistep process, usually with several, if not exclusively non-affinity methods, like ion-exchange, HILIC, size exclusion. You'll get deep into the biochemistry of your protein and its various domains to come up with this. Optionally you'll have an affinity step with a tag that you actually want to leave on your protein for pharmaceutical reasons, like Fc, if it's secreted. Considerations like that almost never leave a his-tag in the running for very long.
There is one approved drug with a His tag on it - Blincyto from Amgen. Although this would seem like a good precedent, the approval was partly based on Blincyto having extremely low doses and it being a late-stage (effectively end-of-life) cancer drug. For other drugs, it is generally assumed that a His tag is a non-starter for the FDA, but clearly there is at least this one exception. Although it is true that little data on His tag immunogenicity exists, the view of the FDA is that His tag antibodies exist (they are commercially available), and eventually the human immune system will notice even the smallest non-human proteins. This is based on the observation that about a third of patients taking animal-derived insulin eventually developed allergies, despite the animal insulin being nearly identical to human.
I don't want to sound like an advertisement, but there is a self-removing tag system available from Protein Capture Science (www.proteincapturescience.com), which provides a tagless target. The tag is bound by the proprietary resin, and is induced to release the tagless target from the resin by a shift in pH from 8.5 to about 6.5 (the tag self-cleaves and remains bound to the resin). The resin is also highly reusable, where a simple acid wash strips off the cleaved tag before reuse. The results are pretty impressive, especially with mammalian-expressed proteins.
Full disclaimer - I am in the inventor of the technology and a co-founder of the company. The good news is that the technology is commercially available, and we usually ship the same day as the order. And yes, we have highly established partners who are ready to make a validated resin for GMP applications as soon as there is demand. You can reach us through the web page, and there are examples and manuals and sample plasmid maps there for download. It is also pretty easy to try out, and I am happy to talk more if you are interested.
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