*** GH Testing ***

[Sgt. Hartman]

There's always a lot of talk on here about people wanting to get their GH tested and recently another board has posted results of several generic GH brands that they paid to have tested at a licensed US facility.

The problem with giving these tests any credibility is that there currently isn't a proper method of testing a recombinant protein based drug for "biological activity". In other words it could test to be pure 191aa GH and have absolutely none of the desired effect when administered. This article is written by Boris Y. Zaslavsky, Department of Physiology and Biophysics, Cornell University Medical College.




Recombinant DNA (rDNA) technology has led to the development of new protein-based drugs that are gaining worldwide regulatory approval. Human growth hormone , human insulin , β- and γ-interferons, and erythropoietin are just a few examples of approved rDNA-derived biopharmaceuticals.

The biological effects, purity, and potency of a drug is governed by the chemical structure of the drug for both traditional drugs and biopharmaceuticals. Standard analytical methodologies used for structural analysis of conventional drugs are, however, inadequate for complete characterization of protein-based products.

Two main reasons for this inadequacy are the large molecular size and conformational flexibility of protein-based drugs. The large molecular size hinders the possibility to detect, for example, repla***ent or chemical modification of a single amino acid residue or a change in a single glycosylation site. These alterations of the biomolecule structure, however, may lead to subtle changes of the molecule conformation resulting in significant changes in the pharmacological properties of the product.

Additionally, the wrong choice of manufacturing conditions or formulation may lead to improperly folded polypeptide chains which are biologically inactive. Hence, further methodologies capable of analysis of the protein conformation are needed.

Currently, the analysis of biopharmaceuticals relies heavily on the use of sophisticated methods for the demonstration of the structural identity, homogeneity and purity of the products. These methods include amino acid and carbohydrate analysis, N- and C-terminal sequence analysis, spectroscopic (UV, CD, ORD) analysis, peptide mapping, electrophoresis, chromatographic purity profile methods, potency/activity assays, etc. It must be emphasized that no one method is considered to be sufficient in itself, and that multiple methods are necessary to completely characterize and/or control such products.

For example, amino acid analysis for proteins with molecular weights above about 16 kilodaltons is known to be of very limited value. While useful for identification of the target protein, N-terminal and C-terminal sequencing only partially characterize the protein. Analysis of the primary structure, however, is insufficient to assure the biological potency of a protein, particularly since the potency depends on the protein conformation.

The conformation of proteins is usually analyzed by optical spectroscopy, such as UV spectroscopy, fluorescence spectroscopy, optical rotary dispersion, or circular dichroism. These methods are generally not sensitive enough to detect the subtle conformational changes caused by small alterations in the protein structure, especially if these changes do not affect side-chain chromophores from tryptophan, phenylalanine, tyrosine, and cysteine residues within the protein. Furthermore, these methods as well as others, such as electrophoresis, isoelectric focusing, differential scanning calorimetry, light scattering, ultracentrifugation, gel filtration, and immunological assays, only provide information about a particular structural or functional feature of a protein.

Chromatography is currently the most widely used analytical method for determining the purity of small organic drugs. Four modes of High-Performance Liquid Chromatography (HPLC) currently used for protein analysis are size-exclusion, ion-exchange, reversed-phase, and hydrophobic interaction chromatography. All these HPLC methods, though commonly employed to monitor the purity of biopharmaceuticals, are usually incapable of resolving proteins that differ by one or two residues or detecting other small changes in the macromolecular structure.

Hence, while chromatography is sufficient for determining whether a small organic drug is functional, the evaluation of a biopharmaceutical requires measurements of biological activity. Many of these measurements are the animal-based assays, particularly when the mechanism of action of the biopharmaceutical is not well defined. These assays are generally imprecise (with variability often 30% to 100%), time-consuming, and costly, and are not rugged. Cell culture assays can be used when the protein-based drug produces a measurable response in a cell-based system. The variability of these assays is much lower, often in the range of 10% to 30% or better (e.g., in vitro clot lysis assay has a variability of about 5%).

Physicochemical tests are much faster, more precise, and more reliable than biological assays. A physicochemical test providing information related to the biological potency of a protein-based drug would improve the control of the safety and efficacy of the drug.

Such a test should meet the following requirements: (1) it should provide information quantitatively related to the biological potency of a biopharmaceutical, (2) it should be capable of detecting minor changes in the structure of large macromolecules, (3) it should be especially sensitive to the structural changes affecting the efficacy of a biomacromolecule, (4) it should be sensitive to the presence of impurities in the product in quantities as small as 0.1 to 0.01 wt. %, (5) it should be simple, precise, and rugged, and (6) it should be time-, labor-, and cost-effective so as not to increase the overall cost of the product. Even if only some of these requirements were met, the test would improve the possibilities for assuring the safety and efficacy of biopharmaceuticals, such as recombinant human growth hormone (rhGH).

[gixxerboy1]

great post

[ScotchGuard02]

Thanks for the info.

[ramacher]

Just to be certain, why risk getting bunk gh? Most of the generics ARE crap and way over priced (even higher than one pharm brand I can think of), but to be sure just stick to pharmacy grade with gh. You have some room for error with AAS, especially if you home brew, but you can never be sure what is in those little vials, green tops, blue tops, whatever, doesn't matter.

[lovbyts]

Just to be certain, why risk getting bunk gh? Most of the generics ARE crap and way over priced (even higher than one pharm brand I can think of), but to be sure just stick to pharmacy grade with gh. You have some room for error with AAS, especially if you home brew, but you can never be sure what is in those little vials, green tops, blue tops, whatever, doesn't matter.

It always boils down to $$$$ People dont always want to or have the money to spend on pharmacy grade or have access. Many are also mislead to think what they are getting is pharmacy grade were in fact it's nothing more than China bunk IGF or worse.

[baseline_9]

Nice....

[marcus300]

Its what ive been trying to say all along

Good thread lets keep it at the top

[Sgt. Hartman]

Bump

[DanB]

so basically the only way is to have igf levels done then yeah?

[Sgt. Hartman]

Not really, because you could be injecting IGF-1 which would also give an elevated test result.

[DanB]

ah yes true enough, but then again I seen alot of testing to show bunk igf aswell haha

so they is no real test then, exceot from the results themselves that is, correct?

[Sgt. Hartman]

IMO testing GH serum 4hrs after pinning and testing igf is the best testing method but still not fool proof. With pharm grade the results should speak for themselves.

[jimmyinkedup]

Good post Sgt.....

[Fit N Fun]

Since HGH is metabolised into IGF-1 and IGF-1 is easy and relatively cheap to have tested, it is therefore easy to establish whether your HGH is legit.

Did not find anyone able to test for serum HGH in my country, but the IGF-1 test I used was a blood spot test and mailed to a lab in the USA.

I think its OK to talk about where you get testing done, so some more info on this would be good

[gixxerboy1]

Since HGH is metabolised into IGF-1 and IGF-1 is easy and relatively cheap to have tested, it is therefore easy to establish whether your HGH is legit.

Did not find anyone able to test for serum HGH in my country, but the IGF-1 test I used was a blood spot test and mailed to a lab in the USA.

I think its OK to talk about where you get testing done, so some more info on this would be good

what if you are injecting igf instead of gh. Your not going to be able to tell.

[run_n_fool]

There's always a lot of talk on here about people wanting to get their GH tested and recently another board has posted results of several generic GH brands that they paid to have tested at a licensed US facility.

The problem with giving these tests any credibility is that there currently isn't a proper method of testing a recombinant protein based drug for "biological activity". In other words it could test to be pure 191aa GH and have absolutely none of the desired effect when administered. This article is written by Boris Y. Zaslavsky, Department of Physiology and Biophysics, Cornell University Medical College.




Recombinant DNA (rDNA) technology has led to the development of new protein-based drugs that are gaining worldwide regulatory approval. Human growth hormone , human insulin , β- and γ-interferons, and erythropoietin are just a few examples of approved rDNA-derived biopharmaceuticals.

The biological effects, purity, and potency of a drug is governed by the chemical structure of the drug for both traditional drugs and biopharmaceuticals. Standard analytical methodologies used for structural analysis of conventional drugs are, however, inadequate for complete characterization of protein-based products.

Two main reasons for this inadequacy are the large molecular size and conformational flexibility of protein-based drugs. The large molecular size hinders the possibility to detect, for example, repla***ent or chemical modification of a single amino acid residue or a change in a single glycosylation site. These alterations of the biomolecule structure, however, may lead to subtle changes of the molecule conformation resulting in significant changes in the pharmacological properties of the product.

Additionally, the wrong choice of manufacturing conditions or formulation may lead to improperly folded polypeptide chains which are biologically inactive. Hence, further methodologies capable of analysis of the protein conformation are needed.

Currently, the analysis of biopharmaceuticals relies heavily on the use of sophisticated methods for the demonstration of the structural identity, homogeneity and purity of the products. These methods include amino acid and carbohydrate analysis, N- and C-terminal sequence analysis, spectroscopic (UV, CD, ORD) analysis, peptide mapping, electrophoresis, chromatographic purity profile methods, potency/activity assays, etc. It must be emphasized that no one method is considered to be sufficient in itself, and that multiple methods are necessary to completely characterize and/or control such products.

For example, amino acid analysis for proteins with molecular weights above about 16 kilodaltons is known to be of very limited value. While useful for identification of the target protein, N-terminal and C-terminal sequencing only partially characterize the protein. Analysis of the primary structure, however, is insufficient to assure the biological potency of a protein, particularly since the potency depends on the protein conformation.

The conformation of proteins is usually analyzed by optical spectroscopy, such as UV spectroscopy, fluorescence spectroscopy, optical rotary dispersion, or circular dichroism. These methods are generally not sensitive enough to detect the subtle conformational changes caused by small alterations in the protein structure, especially if these changes do not affect side-chain chromophores from tryptophan, phenylalanine, tyrosine, and cysteine residues within the protein. Furthermore, these methods as well as others, such as electrophoresis, isoelectric focusing, differential scanning calorimetry, light scattering, ultracentrifugation, gel filtration, and immunological assays, only provide information about a particular structural or functional feature of a protein.

Chromatography is currently the most widely used analytical method for determining the purity of small organic drugs. Four modes of High-Performance Liquid Chromatography (HPLC) currently used for protein analysis are size-exclusion, ion-exchange, reversed-phase, and hydrophobic interaction chromatography. All these HPLC methods, though commonly employed to monitor the purity of biopharmaceuticals, are usually incapable of resolving proteins that differ by one or two residues or detecting other small changes in the macromolecular structure.

Hence, while chromatography is sufficient for determining whether a small organic drug is functional, the evaluation of a biopharmaceutical requires measurements of biological activity. Many of these measurements are the animal-based assays, particularly when the mechanism of action of the biopharmaceutical is not well defined. These assays are generally imprecise (with variability often 30% to 100%), time-consuming, and costly, and are not rugged. Cell culture assays can be used when the protein-based drug produces a measurable response in a cell-based system. The variability of these assays is much lower, often in the range of 10% to 30% or better (e.g., in vitro clot lysis assay has a variability of about 5%).

Physicochemical tests are much faster, more precise, and more reliable than biological assays. A physicochemical test providing information related to the biological potency of a protein-based drug would improve the control of the safety and efficacy of the drug.

Such a test should meet the following requirements: (1) it should provide information quantitatively related to the biological potency of a biopharmaceutical, (2) it should be capable of detecting minor changes in the structure of large macromolecules, (3) it should be especially sensitive to the structural changes affecting the efficacy of a biomacromolecule, (4) it should be sensitive to the presence of impurities in the product in quantities as small as 0.1 to 0.01 wt. %, (5) it should be simple, precise, and rugged, and (6) it should be time-, labor-, and cost-effective so as not to increase the overall cost of the product. Even if only some of these requirements were met, the test would improve the possibilities for assuring the safety and efficacy of biopharmaceuticals, such as recombinant human growth hormone (rhGH).

Great post sgt. Question for you, would one experience typical sides on improperly folded peptides? Assume in this case serum hgh test was affirmative ...

[Sgt. Hartman]

I wouldn't think so as it would be "biologically inactive". But a lot of people think that they've put anti diuretics and other chemicals in chinese generic GH to mimic the sides.

[Fit N Fun]

what if you are injecting igf instead of gh. Your not going to be able to tell.

I also tested my HGH using mass spectroscopy at a university lab to show it was pure 191AA, this does not prove it is active, which is where the IGF-1 test completes the picture.

[DanB]

I shall bump this thread,

I'm sure most have seen the serum results for a certain batch of rips on certain boards from a certain private source that I'm sure most here know of (dont pm to ask for source, I wont reply)

Three (mabey more but I havnt seen them) of these results have abnormally high levels, 67ng/ml and the other two is something stupid like 110-115ng/ml, these all claim to be taken using standard protocol for serum test

I dont believe these to be the true values as I have seen legit pharm grade come in at less then half those levels and everything I have read indicates that the rips levels arent even possible, yes it varys from person to person but these are simply minimum 2x's too high even with the variables.

I doubt the labs are fvcking up the results so frequently and I dont think they are doctored either before they are posted so this leaves us with one reasonable explaniation, and it happens to be the latest ''conspiracy'' theory on the go at the moment

The chinese have found a way to manufacture GH which basically gives stupidly high serum readings but little actual effects or benefits, i.e. biologicaly inactive?

Lately everybody claims to have seen an ''article'' stating this theory but nobody can explain it in depth or even link the article, so does it exist or is this the latest bro science and if it is, then how can the labs be explained?

So my questions are,

1 : Has anybody seen this ''article''?
2 : Is this theory even possible?
3 : If this isnt possible then how can the results be explained? it isnt possible for 10 i.u rips to be 5x's more potent then pharm grade, if you know a bit about GH in general then you will laugh at this statement, Im laughing as Im typing it but this is what some people are trying to claim,

[Sgt. Hartman]

I've been reading the same BS all over the internet, people saying they're testing at 100ng/ml on 10iu. It's ridiculous.

The licensed pharmaceutical companies have done extensive research, and I've posted some of the studies here before, that show while there is a good bit of individual variance, on average, your gh serum 3 - 4 hours after injection should be a little over double the # of iu injected measured in ng/ml. My pharm GH yielded 31.6ng/ml 4 hours after injecting 10iu. So rips are overdosed by 3 - 5 times depending on who you believe?

My personal opinion is that they've found a cheaper or shorter way of making 191aa GH that makes it biologically inactive or at least partially biologically inactive. They make it for so cheap and they're sponsors on several boards and know that everyone is getting these tests done so they overdose the shlt out of it. Yeah so maybe a 10iu vial had 30iu of GH in it but if it has no effect in the body when administered then what's the point. I'd like to see some of these guys IGF-1 levels who are running 3 X overdosed rips lol.

I've never seen this article and I'd suspect that the only people that know what that shlt really is are the ones in charge of producing it.

[Brohim]

I have tested some Rips that the seller said "are so potent don't inject more than a few iu at a time" and it was bunk as hell. Registered like .8 on the serum test. Ive seen most pharma's test in the 30's (10iu then serum test 3.5 hrs. later)

I have not heard of the bio inactive hgh, whatever that means. But IMO you should get a GH Serum test done (50$) and it should register at least around 20 coupled with some type of symptom's.

[Sgt. Hartman]

I have tested some Rips that the seller said "are so potent don't inject more than a few iu at a time" and it was bunk as hell. Registered like .8 on the serum test. Ive seen most pharma's test in the 30's (10iu then serum test 3.5 hrs. later)

That's because rips are garbage plain and simple.

I have not heard of the bio inactive hgh, whatever that means.

Read the first post in this thread.

But IMO you should get a GH Serum test done (50$) and it should register at least around 20 coupled with some type of symptom's.

Which would still prove absolutely nothing if it has no biological effect.

[Fit N Fun]

I have posted on this several times, but it seems like it a hard message to get over.

I used an online lab to carry out a blood spot test for IGF-1 levels, the test was less than $100 so a good investment at a fraction of what I spend on HGH.

The IGF-1 test showed that the HGH I was injecting had increased my IGF -1 levels to 422 ng/ml, I am in my late 50's so will have a natural level of about 140 ng/ml so I know that my body is producing a reasonable response to the HGH I am injecting.

I also had my HGH mass spectrometer tested for purity by a University lab, but this was probably more than was required although a nice to know.

The only way to stamp out the fraudsters is to test, it's great to have the peace of mind to know that you are injecting something that will work.

[marcus300]

Stay away from genetics, they are simply not worth it. Don't be fooled into forum test results when the forum in question is a source board full of fake accounts to push the products with false claims.


The process to produce rHGH is very complicated and the equipment used cost's millions of dollars and many of the generic companies don't have this equipment, this is why to produce rHGH it costs a lot of money due to the expensive equipment need to complete the process. I do know someone who I am in contact with and in one of his first emails to me he describes the process of testing this horomone. I will copy the email so you get more of an understanding. My contact has many titles, he is a Professor of research, Scientific manager, he is head of the Biopharmaceutical Bioprocessing technology centre, Director of Mass spectrometry in clinical pharmacology who provide protein analysis services to commercial and academic clients and also a Doctor



Hi Marcus,


there are some things that we can do, some things that we could do, but that would possibly be too expensive (not worth it) and other things that we cannot do.


Please let me start with the last section:


We absolutely cannot test any substances for their suitability for any purpose, particularly not for their use in a diagnostic, therapeutic and/or recreational purpose, when this invloves administering to a living being (including humans).


Measuring the concentration of the growth hormones is something that is actually not as easy as it might seem. The concentration of the hormone can have two different meanings, it could be the chemical concentration of a compound (this is something that we can measure), it could also be the biological activity of this compound (this would be different, if a certain proportion of this chemical would be biologically inactive, which could happen for a variety of reasons). The latter (measuring the biological activity) is something we cannot do. We can measure the chemical concentration of human growth hormone (or any other growth hormone ), but that would involve the chemical synthesis of an internal standard, followed by a fairly complicated experiment, in which we use chemical scissors (en enzyme called trypsin) to chop the protein into pieces, then we analyse these pieces using a combination of high performance liquid chromatography and mass spectrometry, followed by a computer-intensive analysis of the data. The result of this experiment would be the concentration either in units of mol/l or in units of mg/l (the two are interconvertible). I do not know how the IU (stands for International Units) for growth hormone are defined - this might be a functional (biological) unit (which we cannot measure).


Human growth hormone is a chain of 191 different amino acids. One thing we can do is measure the total molecular weight of the protein in a sample to see, if it corresponds to the molecular weight that would be predicted for a protein containing these 191 amino acids. This experiment detects, if one amino acid was missing or another one was added, in some cases (but not in all cases) even, if an amino acid was replaced with a different one. Another outcome of this experiment would be to see, if other, similar proteins are present in the same sample.


In a different experiment we can check, if a protein in a particular sample is really growth hormone or if it is possibly something completely different, for example egg white protein or milk protein .


For your information, I attach two links to articles about human growth hormone :



Best Wishes