Calm Logic
GLP-1 Specialist
HGH is a peptide too but it so large that it is a protein, which is many times more likely to denature. So people on Meso would be the canary in the coalmine, and they are fine.
All right, i searched for freezing tirz, specifically the Jano response but...
- Thread starter Gr33dyOctopus
- Start date
That is a good general point! But peptides individuals can be very weird, in potentially being super stable or super unstable due to the amino acid residues and the folding structure stability.
Calm Logic
GLP-1 Specialist
That is possible but unlikely when HGH is so fragile in comparison.
Gr33dyOctopus
GLP-1 Enthusiast
Gr33dyOctopus
GLP-1 Enthusiast
Everything I've ever read about this reccomends keeping bac water at room temp! Even tho it seems like it would make more sense to put it in the fridge at least.
Gr33dyOctopus
GLP-1 Enthusiast
Yep, thanks to Calm Logic im not filtering, tho I bought fucktons of filters. At some point maybe, but not convinced I need to, and yes risk tolerance is ramping up.since I've been here.
I think freezing reconstituted glp1’s is currently transitioning from being not advised to being seen as generally ok. That is why there is so much conflicting information out there. Just taking the pulse of this community and a few others, with the leaks from the test groups (I’m not a member). Generally advised if you do it, only do it once (one thaw cycle).
I personally see it as, which would I rather risk, a vial that is potentially destroyed from freezing, and doesn’t do anything, or a vial that is potentially contaminated because I kept it around 3 months instead of 1 (I’m talking greys here not compounded). I’m going to prefer to risk injecting something destroyed vs risking a contaminated vial every time. And the stuff is so cheap if I inject it and nothing happens I’ll just buy more.
I personally see it as, which would I rather risk, a vial that is potentially destroyed from freezing, and doesn’t do anything, or a vial that is potentially contaminated because I kept it around 3 months instead of 1 (I’m talking greys here not compounded). I’m going to prefer to risk injecting something destroyed vs risking a contaminated vial every time. And the stuff is so cheap if I inject it and nothing happens I’ll just buy more.
Calm Logic
GLP-1 Specialist
From a 2015 study of a GLP frozen in human plasma: "Freeze–thaw cycles did not significantly affect stability of GLP-1 or glucagon."
Though human plasma is far more protective than BAC or saline BAC, it gives me the idea that it may be a little better to recon tirz with saline BAC if freezing later. (Reta is said to become cloudy sometimes with saline BAC.)
I found the study just now, posted two months ago at Reddit:
Though human plasma is far more protective than BAC or saline BAC, it gives me the idea that it may be a little better to recon tirz with saline BAC if freezing later. (Reta is said to become cloudy sometimes with saline BAC.)
I found the study just now, posted two months ago at Reddit:
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Nathanologist
GLP-1 Enthusiast
It is aggregated second hand data. It’s not perfect, but majority rules IMO until sound first hand data is available.
Nathanologist
GLP-1 Enthusiast
Next time toss them into my mailbox…
Most of this is correct. I'll take a deep dive into this subject that will likely bore most, but be interesting to the highly-pedantic folks here who really do want to get to the bottom of things.
In chemistry you're taught that reaction rate kinetics are highly temperature-dependent and a small drop in temperature can easily cut reaction rate in half. Many will try to extend that thinking to biological systems, but while often true, that's not strictly correct. Try making yogurt and you'll find that different bacterial cultures thrive in different temperature ranges. Most of the common ones thrive around 100F, which makes perfect sense if you think about it, since these cultures typically had to evolve to optimize themselves at mammalian body temperature. At the same time, you have varieties (like Filmjolk) that do better at room temperature, which evolved in milk left out on the countertop.
Most (but not all, as you correctly note) bacteria will grow more slowly at refrigerator temps and all will stop growing below freezing (since life is water-dependent).
I would disagree with some of the claims made elsewhere about the benzyl alcohol freezing at a different temperature than water and the need to mix against after a freeze-thaw cycle. As a pure compound the freezing temperatures are different, but as a solution, that's not the right way to think about it. I'll unpack that a bit for you:
At higher concentrations of benzyl alcohol in water, you'd experience "fractional freezing" as the temperature dipped below 0C. That's to say that the temperature would vary across a range, as freezing occurred. The very first bit of ice to form would likely be richer in water and weaker in alcohol (vs the solution as a whole) and the very last bit of ice to form would likely be richer in benzyl alcohol (vs the solution as a whole). It's tempting to imagine that the water freezes first (at its freezing point) and then the benzyl alcohol freezes later (at its freezing point), but that's not correct. It freezes across a temperature range. Lookup fractional freezing for more details. It's analogous to distillation, only happening at the freezing point rather than the boiling point.
However, when you have a weak solution like this (just under 1% benzyl alcohol), you don't even need to consider the complexity of fractional freezing. The simplified gen chem concept of "freezing point depression" will be sufficient to analyze the freezing behavior and you can pretend that the whole solution freezes at once at a temperature slightly below 0C.
I can say that if you did choose to freeze BAC water, I wouldn't expect there to be a benefit to deep freezing vs freezing. Once you've frozen it, you've already stopped bacteria from growing and all deep freezing is going to do is introduce additional strain on the structural components of the container due to the thermal expansion properties varying across the different materials used to seal the container VS the glass itself.
My personal opinion is that freezing is generally unnecessary (but shouldn't hurt either), unless you're planning on keeping it for many years. An exception to this would be if I had already started using a container of BAC water and then decided I wasn't going to use that container any more for the next year. In theory, by using the container you could have introduced bacterial contamination to the BAC water, which the alcohol should kill, but in the edge case where it somehow didn't (and could feed off of some material inside of the container), you'd freeze additional growth of that bacteria until you thawed the container again. Then again at ~$10 each, I'd probably just chuck it and start with a new one in that situation. That also means I wouldn't freak out about receiving a shipment of BAC water that froze in transit (which I'm sure is giving plenty of people anxiety right now and leading to some people paying extra for overnight shipping).
Regarding the instructions to keep at room temperature VS refrigerating: I would take that to mean the manufacturer rigorously validated the results for the product being held at room temperature throughout storage and use and found that it performed as desired when stored and used that way. I don't think this means they tested refrigeration and ran into problems when they did, but rather that they simply chose not to make their testing more complicated (and expensive) than it needed to be. Until someone can present a plausible reason why refrigerating BAC water would be expected to have a negative impact, it's hard to dig deeper into this controversy. Both refrigeration as well as storing in an opaque container that stops light from getting through would theoretically be expected to extend the shelf-life based on basic chemistry principles, which I suspect is why optimizers often lean that way.
You also asked "what pH lyophilized tirz will reconstitute at." That's not really the right way of thinking about it unless you're producing it or trying to perform a chemical separation to purify a compound. It's better to think of pH as more of a roadmap than directly causal. For example, let's say I'm adding a particular alkali to force a compound to precipitate out of solution (so that I can filter the solid out to separate it from the liquid). It's not that a high (or low) pH causes it to come out of solution (although some might prefer think of it that way). It's that as I add alkali, the pH will very slowly rise at first (as the reaction is taking place) and then rise more quickly, once I've added enough alkali to push the reaction as far as it's going to go. By keeping an eye on pH, the chemist could infer when they've added enough alkali to complete the extraction, similar to watching a progress bar on a computer.
Oh, yes, this was absolutely riveting! So now predictably I have more questions if you have time to dig into them too. Are you a chemist as your day job? Thanks for putting all this effort into this! It’s really fantastic.
Yes great point I’d not really fully connected to this! And I’d always thought that might be because enzyme configuration or specific temperature dependent fluidity enables or obstructs presentation of necessary substrates ar different temperatures. But the mechanism must be very dynamic and clever. (Like heparin/heparan).
Ugh Listeria will literally walk/flow around and invade crevices. And is all over our food supply chain. Cover and seal.
Ah thank you, new concept. Yes, I’ve seen this when slowly freezing a slushy. Okay, like how an alcohol and sugar slushy acts when freezing down. So the species don’t to be be 100% miscible to show this behavior? And so the outcome for us is… freeze it, no harm to the dissolved peptides suddenly seeing different charge/forces and being destabilized.
Love this analogy, so perfect
Got it. That’s great for the peptide. And bringing the whole solution back to liquid,
Hmmm, so, may I impose on you to talk about freezing a phenol (0.5% and lower) in water solution? I know there’s 2 eutectic points, and some phenol hydrate, which I don’t actually understand, but pertaining to peptides, lots of compounders use phenol not benzyl alcohol so would freezing impact the future phenol solution itself, and would that harm the peptide (tirz has a reputation of super stability but even so). Also why do compounders use 0.5% phenol when Eli Lilly only uses 0.18%?
New distraction, why, mechanistically, do labs have -80 freezers? There is some atomic motion / oxidation / chemical reactions left, and for living creatures survival is better lower? Is it because of their enzymes and proteins still are [slowly] aging?
Benzyl alcohol itself oxidizes with so many influences. Oxygen, alkaline pH, presence of benzaldehyde, it’s oxidation product, UV exposure (fluorescent lighting), warmth. I think it only lasts 28 days at fridge especially because room air enters once you withdraw enough from the bottle. So I think I might be be cheap and freeze some BAC for later use before they reach their use by date and if I only use 2ml after opening. Thank you for sorting out what is happening.
I know the hospira plastic (propylene-ethylene) will become brittle below -20, and the butyl rubber stopper too probably below -40,
👍. Benzyl alcohol is not really so effective, fairly limited spectrum and for instance a mediocre disinfectant with 3 log reduction for staph aureus, one of the more likely contaminants. Good thing for immune systems.
👍
Someone said there were complaints of a precipitant at refrigeration temp that had to be redissolved. (Gah, should always be thrown away by healthcare workers at that level). My theory is they could have been seeing precipitated benzoic acid (secondary oxidation product after benzaldehyde is what I’d read, with just enough different solubility between fridge and room temp to cause precip if you had even 0.3% benzoic acid ) so complainants could have been seeing an aged out solution that should have been thrown away.
Opaque containers would probably help. 0.9% is already marginally effective. I haven’t been able to find real data showing bacteriostatic efficacy as a function of concentration, 1.1% seems to be agreed to be really good, less than half may be so useless that they don’t study it? 2.0% starts to stress delicate proteins like some antibodies
Oooh my brain is feeling strain lol. Thank you for another new idea. Yeah that was the contrst I’d read about those ideas.
Ah. I had only read about pH tugging on the charges of the amino acids and helping them ionically join the solution. So depending on the identities solubilizing might be better at different pHs.
Mostly though my thinking was/is basically a confused mishmash. A little bit about solubility, some about bound TFA and whether that remains as a contaminant (or if we really were given research grade with it as a major component - what is that overfill, hmmm?) How do we know if the tirz is partnered with TFA, acetate, or sodium
And some about what actually happens… in the bloodstream and interstitial spaces with their pH? Do these forms all revert to their base form or the same form in us? Are we near an isoelectric point? What did the FDA think was studied? And in what ways does any of this matter (well, toxicity, maybe timing and relative affinity to various receptor variants.) Please cast some light, professor ☺️.
Don't do much with chemistry these days, but obtained the education about 25 years ago, so bits and pieces of it still bounce around in my brain. I can't necessarily answer specific questions definitively, but can speculate or point out well-established principles that relate to the topic.
To be clear, I was only talking about freezing BAC water and not solutions of peptides and other things in BAC water. When you move from simple compounds to more complex organic molecules and structures, it's more complicated and usually just best to see if someone has tested the case you're interested in rather than trying to opine from first principles. Some compounds will be unaffected by freezing or freeze/thaw cycles, while others will degrade or undergo other changes.
Freezing a peptide dissolved in water is very different from keeping a freeze dried peptide at the same temperature. When water freezes, it stops being a fluid and locks into a solid crystal structure, in addition to expanding in volume. That can potentially be highly damaging to materials in the water. Potatoes are a good example of this in that even after boiling them (which damages/cooks lots of cells), freezing them damages the cells on a whole different level. Try freezing and then thawing your favorite potato meal and observe the change that takes place.
Anecdotally, many GLP1 fans have done okay freezing and thawing certain reconstituted peptides, but that's going to be case by case for different compounds and won't generally be true of all. This is why the rule of thumb is to keep peptides freeze dried for storage and only add water at the point of use.
In regards to the physical components (e.g. plastic, rubber), it's a combination of becoming brittle at lower temperatures and thermal expansion effects. For example, above a certain temperature a tight seal would be maintained between the glass and rubber, similar to a cork in a bottle. As the temperature decreases the rubber becomes more rigid, locking into whatever position it was in before the temperature was brought down. As the temperature continues to decrease, it's going to depend on how thermal expansion varies between the glass vial and the rubber stopper. If the size of the rubber reduces more quickly than that of the glass, you've just created a small gap between the rubber and the glass. At such low temperatures you probably don't have bacteria creeping in, but over a really long period of time, the ever so tiny vapor pressure of the compounds could leak out (although this is a REALLY slow effect to the point where I probably wouldn't worry about it). If it turns out the size of the rubber reduces slower than the glass then you're putting extra strain on the glass, which could rupture and fail catastrophically (which should be visually obvious). Those are reasons I might opt not to deep freeze (vs standard freezing), but honestly that concern is probably overkill.
In regards to benzyl alcohol, keep in mind that there are two reasons for it to be added to BAC water. The more obvious reason is what gets discussed here (your reconstituted vial is now more resistant to bacterial growth). The other reason is to prevent cross-contamination (e.g. your BAC water itself can be reused multiple times without itself becoming contaminated).
Regarding pH, keep in mind that it's a measure of the hydrogen/hydronium ion concentration in water, which does factor into some chemical reactions. When I said it was more of a progress bar, what I meant is that pH alone is insufficient to define whether or not something will dissolve, but it very clearly will indicate how far along a reaction taking place in solution has gone. You're correct that oxidation state of amino acids will factor into solubility, which could be inferred from the pH of the solution. But that's more the final pH AFTER adding the compound to the water. Purified water isn't always going to have a pH of 7 (neutral), since it's never truly pure. For example, if you exposure perfectly pure water to air, it will slowly dissolve CO2 from the air, forming carbonic acid and if you had a high-precision pH meter you could actually watch the reading go down slightly over time. I wouldn't obsess over the initial pH of water, since whether the pH is 6 or 8 (due to minor contaminants), the final pH of the water is going to be more dependent on what you're adding to it than what the original value was. Pure water is very touchy and even the smallest addition can swing the pH more than one might expect. If you're working with a compound that was to require a specific pH for proper solubility, presumably they'd either mix something in with the compound to ensure that pH is achieved after it's added to water or they'd instruct you to add it to something other than pure water that allows the end product to achieve the desired pH. Or other options to increase solubility of less soluble compounds could be varying temperature (typically heating) or increasing the amount of water/solution, since if you have twice as much water, you can typically dissolve twice as much stuff in it before it becomes saturated.
Regarding body pH, you'd want to read up on the concept of "buffering." Your body is constantly doing that to ensure the pH of blood and other fluids remain in the desired range. This is why you don't have to sweat the pH of each individual item you're eating or drinking and your body can still keep blood pH where it wants. When it comes to injecting stuff into your body, that's a little different since your body has less defenses against that. It can handle variations within reason using similar tricks.
Let me put it this way in regards to pH: If you're injecting BAC water with a pH of 2 into your body, the problem isn't so much the pH of 2, but what that implies. In order for the pH to be 2, something has gone horribly wrong in the manufacturing process and a strong acid found its way into your bottle somehow. In fact, I'd assume checking the pH of BAC water batches at the production facility is going to be one of several QC checks in the process. If you as an individual are having to worry about the pH of something discussed on this forum, that means something upstream in the supply chain has went horribly wrong, since that should all have been worked out before getting to you.
To be clear, I was only talking about freezing BAC water and not solutions of peptides and other things in BAC water. When you move from simple compounds to more complex organic molecules and structures, it's more complicated and usually just best to see if someone has tested the case you're interested in rather than trying to opine from first principles. Some compounds will be unaffected by freezing or freeze/thaw cycles, while others will degrade or undergo other changes.
Freezing a peptide dissolved in water is very different from keeping a freeze dried peptide at the same temperature. When water freezes, it stops being a fluid and locks into a solid crystal structure, in addition to expanding in volume. That can potentially be highly damaging to materials in the water. Potatoes are a good example of this in that even after boiling them (which damages/cooks lots of cells), freezing them damages the cells on a whole different level. Try freezing and then thawing your favorite potato meal and observe the change that takes place.
Anecdotally, many GLP1 fans have done okay freezing and thawing certain reconstituted peptides, but that's going to be case by case for different compounds and won't generally be true of all. This is why the rule of thumb is to keep peptides freeze dried for storage and only add water at the point of use.
In regards to the physical components (e.g. plastic, rubber), it's a combination of becoming brittle at lower temperatures and thermal expansion effects. For example, above a certain temperature a tight seal would be maintained between the glass and rubber, similar to a cork in a bottle. As the temperature decreases the rubber becomes more rigid, locking into whatever position it was in before the temperature was brought down. As the temperature continues to decrease, it's going to depend on how thermal expansion varies between the glass vial and the rubber stopper. If the size of the rubber reduces more quickly than that of the glass, you've just created a small gap between the rubber and the glass. At such low temperatures you probably don't have bacteria creeping in, but over a really long period of time, the ever so tiny vapor pressure of the compounds could leak out (although this is a REALLY slow effect to the point where I probably wouldn't worry about it). If it turns out the size of the rubber reduces slower than the glass then you're putting extra strain on the glass, which could rupture and fail catastrophically (which should be visually obvious). Those are reasons I might opt not to deep freeze (vs standard freezing), but honestly that concern is probably overkill.
In regards to benzyl alcohol, keep in mind that there are two reasons for it to be added to BAC water. The more obvious reason is what gets discussed here (your reconstituted vial is now more resistant to bacterial growth). The other reason is to prevent cross-contamination (e.g. your BAC water itself can be reused multiple times without itself becoming contaminated).
Regarding pH, keep in mind that it's a measure of the hydrogen/hydronium ion concentration in water, which does factor into some chemical reactions. When I said it was more of a progress bar, what I meant is that pH alone is insufficient to define whether or not something will dissolve, but it very clearly will indicate how far along a reaction taking place in solution has gone. You're correct that oxidation state of amino acids will factor into solubility, which could be inferred from the pH of the solution. But that's more the final pH AFTER adding the compound to the water. Purified water isn't always going to have a pH of 7 (neutral), since it's never truly pure. For example, if you exposure perfectly pure water to air, it will slowly dissolve CO2 from the air, forming carbonic acid and if you had a high-precision pH meter you could actually watch the reading go down slightly over time. I wouldn't obsess over the initial pH of water, since whether the pH is 6 or 8 (due to minor contaminants), the final pH of the water is going to be more dependent on what you're adding to it than what the original value was. Pure water is very touchy and even the smallest addition can swing the pH more than one might expect. If you're working with a compound that was to require a specific pH for proper solubility, presumably they'd either mix something in with the compound to ensure that pH is achieved after it's added to water or they'd instruct you to add it to something other than pure water that allows the end product to achieve the desired pH. Or other options to increase solubility of less soluble compounds could be varying temperature (typically heating) or increasing the amount of water/solution, since if you have twice as much water, you can typically dissolve twice as much stuff in it before it becomes saturated.
Regarding body pH, you'd want to read up on the concept of "buffering." Your body is constantly doing that to ensure the pH of blood and other fluids remain in the desired range. This is why you don't have to sweat the pH of each individual item you're eating or drinking and your body can still keep blood pH where it wants. When it comes to injecting stuff into your body, that's a little different since your body has less defenses against that. It can handle variations within reason using similar tricks.
Let me put it this way in regards to pH: If you're injecting BAC water with a pH of 2 into your body, the problem isn't so much the pH of 2, but what that implies. In order for the pH to be 2, something has gone horribly wrong in the manufacturing process and a strong acid found its way into your bottle somehow. In fact, I'd assume checking the pH of BAC water batches at the production facility is going to be one of several QC checks in the process. If you as an individual are having to worry about the pH of something discussed on this forum, that means something upstream in the supply chain has went horribly wrong, since that should all have been worked out before getting to you.
My brain just exploded and I gotta go mop it up, but I will be back soon to reply to all this good stuff. TY for the quality discussion! I’m actually all for basic principles, as long as we are aware when they are true. That’s how I like to approach new problems too.
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