myskamuska
New Member
If lyophilised HGH has been in the freezer overnight, is it still fine to use? Do you think it degraded?
MESO-Rx
Anabolic Steroids
Freezing lyophilised hgh
- Thread starter myskamuska
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deadformat83
New Member
This is from Pepchat. They said that it doesn’t tolerate repeated freeze and thaw and that you should just refrigerate. According to this study it degrades some but wouldn’t be unsafe just it loses potency
https://peptide.chatmyskamuska
New Member
So if im able to dissolved it, it should be good to go?
deadformat83
New Member
I would. The only thing is lost potency. The end result said to just refrigerate but nothing about any issue of contamination due to temp or moisture in a vial constituted or not.
PrairieSteel
New Member
Sounds like it has denatured. I would discard and chalk it up to a lesson learned
I think this test is for reconstituted HGH, not lyophilized. Don’t see any reason why a lyophilized peptide would degrade after freezing.
Ghoul
Member
There is something called the “glass transition temperature”, and it’s not water freezing temp, but lower, and depends on the formulation of the lyophilized peptide.
“ Glass transition temperature (Tg) is the temperature where an amorphous material (like a freeze-dried protein) shifts from hard and glassy to soft and rubbery/mobile.”
Below Tg, molecules are basically “locked in place” and reactions/aggregation happen very slowly.
Above Tg, molecular motion jumps up, so the product can degrade or collapse much faster.”
Every time glass transition temperature is crossed, when the peptide is reconstituted, it shows more damage, mostly taking the form of increased aggregation.
This is why every lyophilized pharm rHGH has a warning to not freeze.
There’s a mountain of evidence supporting this. It’s why -80c is the universally recommended optimal peptide freezing temp by the companies that make them. -80c is far enough below any possible glass transition temp that the normal variations in freezer temp aren’t enough to cause what are effectively “freeze / thaw” cycles to occur. Unlike a household freezer, where the glass transition temp might be crossed a dozen times a day, doing just as much damage as if you put something in the freezer, thawed it, and refroze it many times a day.
This illustration shows the impact on one lyophilized peptide, but the same applies to all. More aggregation, lower biological activity, two aspects of quality the testing available to us via Jano and the other labs are incapable of measuring accurately,
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“It is observed that both F/T and Lyo induce protein aggregation, which in turn causes perturbations in the biological potency of the mAb therapeutic.”
“The primary route of degradation induced by freeze–thawing is protein aggregation.”
Effects of Solution Conditions, Processing Parameters, and Container Materials on Aggregation of a Monoclonal Antibody during Freeze-Thawing
Freeze–thawing is a potentially damaging stress to which therapeutic proteins can be exposed deliberately during storage of bulk drug substance, and a…
“These effects became more extreme when the number of freeze-thaw cycles was increased from 1 to 3.”
“thermal behavior around Tg’, the glass-transition temperature of the excipient-rich phase after freezing, as a function of repeated freeze-thaw cycles.”
DSC reveals the excipient impact on aggregation propensity of pharmaceutical peptides during freezing
Pharmaceutical peptides are susceptible to aggregation in solution, making stabilization by addition of suitable excipients essential. To investigate …
The safest approach, without causing this “glass transition” aggregate inducing degradation, is to store in the coldest part of the refrigerator, or get a cheap -80c cryofreezer.
I spoke to one vendor who figured out their rHGH “cloudy” reconstitution problem was only affecting kits that he stored in the freezer.
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myskamuska
New Member
The initial screenshot detailing the freeze-thaw experiment which was posted by deadformat was conducted on an already reconstituted sample. So you can’t apply its results to lyophilized HGH.There is something called the “glass transition temperature”, and it’s not water freezing temp, but lower, and depends on the formulation of the lyophilized peptide.
“ Glass transition temperature (Tg) is the temperature where an amorphous material (like a freeze-dried protein) shifts from hard and glassy to soft and rubbery/mobile.”
Below Tg, molecules are basically “locked in place” and reactions/aggregation happen very slowly.
Above Tg, molecular motion jumps up, so the product can degrade or collapse much faster.”
Every time glass transition temperature is crossed, when the peptide is reconstituted, it shows more damage, mostly taking the form of increased aggregation.
This is why every lyophilized pharm rHGH has a warning to not freeze.
There’s a mountain of evidence supporting this. It’s why -80c is the universally recommended optimal peptide freezing temp by the companies that make them. -80c is far enough below any possible glass transition temp that the normal variations in freezer temp aren’t enough to cause what are effectively “freeze / thaw” cycles to occur. Unlike a household freezer, where the glass transition temp might be crossed a dozen times a day, doing just as much damage as if you put something in the freezer, thawed it, and refroze it many times a day.
This illustration shows the impact on one lyophilized peptide, but the same applies to all. More aggregation, lower biological activity, two aspects of quality the testing available to us via Jano and the other labs are incapable of measuring accurately,
View attachment 365138
——-
“It is observed that both F/T and Lyo induce protein aggregation, which in turn causes perturbations in the biological potency of the mAb therapeutic.”
“The primary route of degradation induced by freeze–thawing is protein aggregation.”
Effects of Solution Conditions, Processing Parameters, and Container Materials on Aggregation of a Monoclonal Antibody during Freeze-Thawing
Freeze–thawing is a potentially damaging stress to which therapeutic proteins can be exposed deliberately during storage of bulk drug substance, and a…www.sciencedirect.com
“These effects became more extreme when the number of freeze-thaw cycles was increased from 1 to 3.”
“thermal behavior around Tg’, the glass-transition temperature of the excipient-rich phase after freezing, as a function of repeated freeze-thaw cycles.”
DSC reveals the excipient impact on aggregation propensity of pharmaceutical peptides during freezing
Pharmaceutical peptides are susceptible to aggregation in solution, making stabilization by addition of suitable excipients essential. To investigate …
The safest approach, without causing this “glass transition” aggregate inducing degradation, is to store in the coldest part of the refrigerator, or get a cheap -80c cryofreezer.
I spoke to one vendor who figured out their rHGH “cloudy” reconstitution problem was only affecting kits that he stored in the freezer.
In the first paper you mentioned, by Dash et al., the methods section of the paper mention repeatedly freezing and thawing samples in the F/T arm as well as repeatedly reconstituting and lyophilizing the samples in the lyo arm. So again, this can’t be applied to the original poster’s situation at all.
The second paper by Zäh et al. also experiments with repeated freezing and thawing of a solution, which we established above is nothing like the situation the poster is facing.
Not to mention that monoclonals are considerably different than other peptides and require their own considerations.
As for the glass transition temperature of lyophilized HGH, the papers I’ve found regarding the matter, although with different excipients each, both mention the Tg of lyophilized HGH that is devoid of any moisture to be WELL above room temperature, ranging from ~80 Celsius to ~126 Celsius depending on the ratio of HGH to sugars.
1. Solid state chemistry of proteins: I. glass transition behavior in freeze dried disaccharide formulations of human growth hormone (hGH) - PubMed
2. Stability of lyophilized human growth hormone - PubMed
So either way, moving a lyophilized vial of HGH from -20 Celsius to room temperature is nowhere near enough to cross the threshold.
All in all, I would say it’s totally fine to use the lyophilized vial after taking it out of the freezer.
Kralteramon
Member
I freeze all my peptides. I don’t notice any potency loss. Let’s see a test to confirm that potency loss occurs. @Sampei did a test on Lobster HGH to confirm potency loss with and without a vacuum on room temp and frozen after 6 months I recall. No statistically significant degradation occurred. I use frozen HGH and my IGF and GH serum blood tests confirm beyond/ more than the expected response. I wouldn’t worry about potency loss when freezing and thawing a vial.
If you keep it frozen until recon it's fine. You just don't want to freeze it, un-freeze/thaw it and then freeze it again.
Pharma GH specifically says don't freeze but they always play it safe, realistically people have kept GH in the freezer forever and had no issues.
Best option is just get a mini fridge dedicated to peptides and keep it in there.
Ghoul
Member
The commonly made error is assuming pharma-grade rhGH is in any way comparable to UGL rhGH. Real pharma/lab rhGH is carefully formulated and lyophilized with methods validated by moisture/Tg testing, strict drying temp limits to avoid meltback, aseptic conditions, and stored/transported under controlled temps.
UGL setups skip all of that. Even the most basic shit, like sterility and vacuum aren’t guaranteed, so the “normal Tg ranges” from the literature don’t transfer.
We see visible evidence of QA failure constantly, even if most don’t realize it’s not just an aesthetic issue. How often do we see a well formed puck? The common tells of amateur lyophilization are cakes pulled off the vial wall, flakes scattered around the vial (often between the stopper and vial glass), instead of one clean solidly adhered puck that’s standard in pharma vials, no matter how hard they get knocked around. That’s direct evidence of collapse/meltback and uneven drying, leaving wet, protein-heavy amorphous pockets dragging Tg down.
Another huge problem I’ve mentioned before is uncontrolled storage and transport temperatures. Those conditions are enough to drop glass transition temps like a rock, even for pharma, never mind poorly made UGL.
I’m pretty sure no one’s gong to assert exposure to uncontrolled temps in shipping isn’t common.
These vials are shipped and stored without a cold chain, and summer shipping containers can hit 150F+. When a cake gets heated above it’s glass transition temp, it goes rubbery, the cake relaxes and collapses, leftover amorphous material crystallizes faster (mannitol is notorious for this), and that crystallization pushes water into protein-rich pockets. Water plasticizes those pockets and drags glass transition temps even lower, setting up a loop where the next temp cycle of cooling then heating pushes glass transition temps down even further each time.
We see the evidence of this type of damage in anecdotal reports here every day.
Foaming on reconstitution is routinely posted about. It’s a dead giveaway of bad lyo. It means the structure is wrecked, water can’t soak in smoothly, gummy protein-rich zones form, air gets trapped, and stable bubbles form. Those gummy zones are indicative of extremely low-Tg created by poor lyophilization. Pharma doesn’t foam when reconstituted.
Then there’s endless drama here involving instant cloudiness/aggregation right after reconstitution, which users report all the time. Immediate aggregation means the protein spent time in a high-mobility state, basically at some temp well above its Tg, where aggregation ramps fast. And that’s just VISIBLE aggregation.
Janoshik’s testing shows an astonishing rate of lyophilized peptide sterility failures of around 5%. That’s literally hundreds of thousands of times higher than pharma, and it’s probably an undercount. If something as absolutely basic and essential as sterility control is that loose, lyophilization control is loose too.
So when the norm is garbage broken up pucks, foaming, often instant aggregation, meaningful sterility failures, and hot, uncontrolled shipping/storage that actively drives Tg downward, the safe assumption for unknown UGL rhGH is Tg far below the worst-case pharma scenario, easily below −15 °C in the protein-rich pockets. When process and handling are unknown, and clear evidence of shoddy manufacturing failures are norm, not exceptions, the risk-minimizing stance is to assume real-world Tg is closer to worst case scenario, not controlled lab/pharma ideals.
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Ivan221106
New Member
How long can I keep it in the fridge lyophilized?
A dude in the forums found a couple kits he left in his closet for like 3 years, sent it in for testing and the purity had gone from roughly 98% to 96-97%.
GH doesn’t expire like food, the proteins degrade over time. The rate they degrade at depends on storage conditions.
So in the fridge (proper storage) it’ll last a long time. Without proper storage it lasts a long time too, you just lose some purity over time.
