Replies to post #4283 on GTC Biotherapeutics (fka GTCB)

[DewDiligence]

>Thanks so much for the thoughtful reply. It's FAR more stimulating conversation than, "GTC scientists are smart, they'll figure it out".<

Every company who wants to produce FoB’s is going to have to deal with the kinds of issues we’ve been discussing, but you’ve made it sound as though GTC will have unusually high hurdles to jump because its proteins are derived from animals.

Your entire line of discussion comes across to me as belittling GTC’s technology under the guise of asking questions.

My reply about GTC’s intellectual prowess was dead serious—GTC has been thinking about the kinds of issues we’ve been discussing as long as anybody and has some of the best scientists to be found anywhere. Yet your reply to my post was “rolls eyes.” Very revealing.

Here’s an excerpt from #msg-9352093 that’s apropos to our discussion:

>>
Every biologic product is produced somewhat differently, whether it's produced in a human, a goat, a cow, or a traditional bioreactor. They will be produced slightly differently. The advantage of mammalian production is that the basic protein structure itself--what I'm talking about are the amino acids, their sequence, and how they're physically structured--is very much the same. The difference is in what's known as the sugars or carbohydrates--what the scientists like to call the "glycosylation pattern." The glycosylation pattern in each biologic system will be different. The issue isn't, "Is it different?" The answer to that is "Yes." The issue is, "Does it mean anything?"

Now to some degree, there are really two issues that you're concerned about with glycosylation. One is when it's given to a person, will the person's immune system react to it and try to kill it, quote unquote, by having a reaction, or will you end up with a material that doesn't have a sufficient half life [because] it gets cleared out by the liver too fast so that you don't have a practical medicine? For the most part, we've determined that that's not a problem for transgenic mammalian production just as it's typically not a big problem for bioreactor-based materials that have had to face exactly the same issue.

Now, let's assume for a moment that you do run into a glycosylation pattern that's a challenge. Does that mean you can't do it? Well, typically the answer is no. You still have options. One is if we know that there's a glycosylation pattern early on we have some flexibility in making this DNA construct to try to adjust the glycosylation pattern. That's one way to try and address it.

Another way is, after the protein's been made and the mammary gland comes out, we can do what's known as "post-translational modification." That's typically the use of some sort of an enzyme to either add a sugar or snip a sugar off or perhaps snip the entire sugar off so you just have the base protein. And in that way you can attempt to treat the molecule to get to the kind of profile that you need. But the main basic message is that each biologic system does make protein slightly differently in the glycosylation, and that's usually not a showstopper.
<<

The above is from an interview circa 2002. What was true then regarding GTC’s ability to address glycosylation variants ought to be even more true today with five additional years of experience under the company’s belt.

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>Any time you mutate a protein away from its natural sequence, you raise the stakes in the immunogenicity game.<

Actually, glycosylation variants are rarely a cause of immunogenicity. I guess you haven’t followed MAXY, whose raison d’etre is producing such variants deliberately. Immunogenicity is much more likely to be caused of impurities in the manufacturing process, and I addressed this issue from GTC’s perspective in #msg-21459812.

[aslan2772]

Re: Atryn Glycosylation, NXS=>NXT

My appologies if this has already been addresed, I haven't yet caught up on the most recent posts.

Pre_clinical, I think all your points are valid, but you raise possible concerns in #'s 1-3 which are uncommon, worst-case scenarios, IMHO. The exception is point #4, which could be a more realistic concern that I had not thought of.

In particular, the consensus sequences for post-translational modification are well-conserved across species and cell types, so while the sugar content of a given glycosylation site may vary across species, the presumption that a consensus site will be glycosylated is, by definition, not a risky hypothesis. I spent a chunk of my graduate school career mapping out phosphorylation sequences, making point mutants, and studying the underlying signaling of this form of post-translational modification. I also have some experience with glycosylation, ubiquitination, and lipid modification. In my experience, the most interesting sites for post-translational modification are the non-consensus sequences, such as NXS, which tend to be more dynamically regulated in various cell types and species, and are often sensitive to the activation states of particular signaling cascades. That being said, when you change such a site to a consensus sequence, the site is usually (with exceptions of course) fully modified, across a broad range of eukaryotic species and cell types. Thus it makes sense that degree of glycosylation of N135 of antithrombin is variable across expression systems, but the other 3 major glycosylation sites, which are NXT sequences, are not. So, in the case of antithrombin glycosylation at N135, I still think producing fully glycosylated Atryn-"alpha" in goats milk would be a snap -- its just the sugar content of the glysosylation that is likely to be differ from the human form.