08-04-2004, 08:55 PM #1
08-04-2004, 09:31 PM #2
Insulin -like growth factor
The efficient and effective manufacture of recombinant proteins, antibodies, vaccines and viral products in animal cells requires a source of growth factors or some means of growth factor signaling to cells. The traditional method is to provide an external source of growth factors, primarily through the addition of fetal bovine serum to culture media. However, many sectors of the biotechnology
and pharmaceutical industries are now demanding pure recombinant growth factors, made under the highest quality standards, or inclusion in serum-free media formulations.
The insulin-like growth factors (IGFs) were originally discovered and purified from serum. They are considered to be important growth factors for industrial cell culture because:
• They are present in all animal and human sera at concentrations of 100 - 500 µg/L.
• The removal of IGFs from serum can abolish up to 90% of the cell growth-promoting activity.
• They stimulate nutrient uptake, cell growth, protein synthesis and inhibit apoptosis or programmed cell death in a wide range
of cell types.
• Almost all cells have type I IGF receptors, which mediate the biological action of IGFs.
Many industrially important cell types can be cultured in serum-free media that contain high concentrations of insulin (5 - 10 mg/L). This is about 1000-fold higher than the normal physiological concentration of insulin. Insulin only works in cell culture because it acts as a weak substitute for IGFs. Much lower levels of IGFs can replace insulin.
The insulin-like growth factors are structurally related to insulin. There are two forms, IGF-I and IGF-II, which are similar and have closely related actions on cell growth via the same receptor. IGF-I is considered to be the main post-natal growth-promoting factor and IGF-II has major effects during fetal development. IGF-I is a non-glycosylated, single chain polypeptide 70 amino acids in length.
Structure of IGF-I and Insulin
Insulin-like growth Factor-I Insulin
IGF-I is similar in structure to pro-insulin, the precursor of insulin. Pro-insulin is a single chain polypeptide, which is cleaved to remove the connecting C peptide, to form insulin. Insulin has two chains (A and B chain) connected by two disulphide bonds.
The receptors for IGF-I and insulin are also structurally related and both ligands interact with each other’s receptors with very low affinity. In cell culture, the potency of IGF-I is higher than insulin because the cellular responses required for biopharmaceutical production in animal cells are mediated via the type I IGF receptor, not the insulin receptor.
Another important feature is that a family of six IGF binding proteins regulates the biology of IGF peptides. These proteins are found in serum and are also produced by cells in culture. IGF binding proteins bind IGFs with high affinity and generally inhibit the actions of IGFs on cells. This has been exploited by making analogs of IGF peptides that do not bind to IGF binding proteins and are therefore superior to both IGF-I and insulin in cell culture. The most potent of these analogs for commercial cell culture purposes is LongR3 IGF-I.
LongR3I GF-I has been specifically engineered and manufactured by GroPep Limited for use in serum-free cell culture media. Structurally it has two significant modifications — first, one amino acid in the IGF-I structure, the glutamic acid (E) at position 3 has been replaced with an arginine ®, which accounts for the R3 in the name; and second, because the molecule is made as a fusion protein, it has an N-terminal fusion partner which is 13 amino acids long. Thus the “LongTM” in the name.
Structure of LongR3 IGF-I
Replacing the glutamic acid (E) with arginine ® at position 3 is important because this modification significantly reduces the binding of the growth factor to the IGF-I binding proteins, enabling LongR3 IGF-I to be so potent. The addition of the fusion partner also enhances refolding and facilitates high-yield production. The end result is a growth factor 10-fold more potent in cultured cells compared to native IGF-I and 200- to 1000-fold more potent than insulin.
A general comparison of properties related to potency in cell culture is provided in the following table.
LongR3I GF-I is manufactured in genetically engineered E. coli. The manufacturing process uses no animal sourced material, making it regulatory friendly for commercial biopharmaceutical production. The system of production is briefly outlined below:
1. Fermentation. E. coli containing the gene for LongR3 IGF-I are grown in a fermenter. GroPep
Limited’s patented expression system uses inclusion body technology.
2. Homogenization. Bacteria are lysed to release inclusion bodies that are harvested by differential
3. Dissolution. The recombinant fusion protein is released into solution. It is not correctly folded
into its tertiary protein structure at this point.
4. Refolding. The LongR3 IGF-I protein is incubated under controlled conditions so that the
disulphide bonds can correctly form to allow the correct protein structure. The protein would
be biologically inactive or less active in an incorrectly folded form.
5. Purification. A four-step system is used to purify LongR3 IGF-I. This series of steps also incorporates accepted protocols for the removal of bacterial endotoxin.
6. Supply. The product is subjected to quality control assays and is available as a lyophilized
powder or can be manufactured as a liquid for delivery to customers.
It is important to be aware that insulin is acting in cell culture systems as a weak analog of IGF-I.
LongR3I GF-I will therefore work in any serum-free medium or cell culture system in which insulin is used. The potency compared to insulin is best illustrated by the data published by Morris, et al, 20001. They found that LongR3 IGF-I was superior to insulin in terms of recombinant protein production, primarily by increasing the number of viable Chinese Hamster Ovary (CHO) cells in a small production system. LongR3 IGF-I was used in the µg/L range compared to insulin in the mg/L concentration range.
Advantages of LongR3 IGF-I
There are several advantages to using LongR3 IGF-I in cell culture rather than insulin.
1. LongR3 IGF-I is Better Cell Science. Because LongR3 IGF-I acts directly on the type I IGF
receptor it is the right tool for the job. And since far less LongR3 IGF-I is required in media than
insulin it can make downstream processing easier and more efficient as well.
2. LongR3 IGF-I Outperforms Insulin. Published research has shown that LongR3 IGF-I leads to overall greater productivity by increasing cell viability and delaying programmed cell death.
3. LongR3 IGF-I is Readily Available and Regulatory Friendly. Since LongR3 IGF-I is a recombinant protein manufactured in a process without any animal-derived components it eliminates regulatory concerns. It is a proven cell culture product currently employed in the manufacturing process
of a number of FDA-approved biopharmaceuticals. A secure and ample manufacturing capacity
ensures a continual, ready supply for commercial production needs.
4. LongR3 IGF-I is Less Expensive than Insulin. Depending on the amount of LongTMR3IGF-I used to achieve cell growth, and the productivity enhancements one achieves, LongR3 IGF-I can be
significantly less expensive than insulin on a dollar/liter basis as shown on the chart below. Also,
over time, as LongR3 IGF-I usage increases, the cost of production will decrease – making it even
less expensive, while insulin costs have been steadily increasing.
Preparation and use of LongR3 IGF-I
LongR3 IGF-I is supplied as a freeze-dried formulation or as a liquid.
The freeze-dried formulation is packed in an atmosphere of nitrogen at a slight vacuum. To prepare a solution for cell culture, introduce an air filled syringe through the septum to equalize the pressure. Next, add sufficient 10 mM HCl or 100 mM acetic acid to the vial to achieve a peptide concentration of at least 0.1 mg/mL. Concentrations of 1 mg/mL or more are recommended. Mix the solution thoroughly to ensure the peptide is completely dissolved. The solution can then be filtered through a low-protein binding membrane before addition to cell culture medium or it can be added directly to the medium, which can subsequently be filtered.
The liquid product is formulated in acetic acid (100 mM) at a concentration of 5 - 7 g/L and is ready to dilute straight into cell culture medium to achieve a biologically active concentration of about 50 µg/L. The final dilution of 100,000-fold, means that there is no effect on pH or osmolality of the cell culture medium.
A titration for LongR3 IGF-I should be performed for each different application as the optimum concentration may vary slightly depending upon the cell type used and other components present in the medium. The recommended final concentration range of LongR3 IGF-I is 10 to 50 µg/L.
Because LongR3 IGF-I and insulin act through the same cell receptor, the effectiveness of LongR3 IGF-I will be masked if it is added in conjunction with commonly employed concentrations of insulin (~10 mg/L). However, inclusion of physiological levels of insulin (~5 µg/L) in cell culture medium containing the recommended levels of LongR3 IGF-I can result in beneficial synergistic effects in certain applications.
08-04-2004, 09:31 PM #3
IGF1, also known as somatomedin C, is polypeptide hormone about the same size as insulin . It is produced predominantly in the liver in response to growth hormone (GH) release from the pituitary gland. Many of the growth promoting effects of GH are due to its ability to release IGF1 from the liver. The conversion ratio of GH to IGF1 varies greatly in different individuals but most external sources of GH convert around 4-6mcg of IGF per one I.U. of GH. IGF-1 acts on several different tissues to enhance growth. IGF1 belongs in the 'superfamily' of substances known as 'growth factors,' along with epidermal (skin), transforming; platelet derived fibroblast, nerve, and ciliary neurotrophic growth factors. None of the other factors have any bearing on exoskeletal tissue incidentally however These agents all have in common the ability to stimulate cell division, known as mitogenesis, and cell differentiation. Meaning That In the case of IGF1 which does act on muscle tissue it will initiate the growth of new muscle fibers, and subsequently new receptors for testosterone . Users have unanimously concluded that it enhances cycles of steroids significantly. They also seem to be adamant about its ability to reduce fat and improve vascularity a great deal.
The IGF1 Hype
There is a considerable amount of hype surrounding IGF1. Every one is blaming the distended bellies of modern Bodybuilders on it. Also the freaky proportions that old bodybuilders that have been around for years are starting to attain. Anti-aging proponents are touting it as the miracle cure for every thing from Parkinson's disease to Alzheimer's. And the medical community has published numerous articles on it for its ability to cause cancer, diabetes and gigantism. While at the same time performing documented experiments on thousands of patients of muscle wasting diseases. And reporting significant turnabouts in there conditions. So what is a guy to think about IGF1 as far as athletic enhancement is concerned? Well first of all you need to know that most experiments conducted with IGF1 do not list the type of IGF used. I have written Dr. Robert Saline of the Swedish rejuvenation institute on several occasions and we have had in-depth discussions on the subject of IGF1 for physical appearance enhancement. He feels it would be unethical to prescribe IGF1 to a bodybuilder to increase muscle mass simply due to the fact that IGF1 has valid applications in the medical community, (Like I could give a rats ass about "ethical"). He can not argue that it is extremely effective as a promoter of muscle growth far beyond what androgens (steroids) alone can offer. Well fortunately in America IGF1 is not a drug (yet) and the FDA has no control over it as of now. This will change in the very near future however, Im absolutely sure of it.
How to use IGF1
Assuming that you have acquired legitimate IGF1 (R3) long chain, That's IGF1 with the binding protein added. You should take dosages ranging from 60mcg up to 120mcg per day in divided doses. One injection in the morning and again at bed time. Never exceed 120mcg in one day. IGF1 can cause serious gastrointestinal problems such as tumors intestinal swelling diarrhea and vomiting. Most IGF1 comes in a concentration of 1000mcg per ML or CC so it makes it easy to measure in an insulin syringe. 10 IU on the syringe is 100mcg. Do the math.
IGF + Insulin
If you plan on doing IGF1 with Insulin, listen closely IGF1 is not that expensive, sure you can get away with using less by including insulin in the stack, but IGF1 and Insulin together have a pro-insulin effect on your blood sugar balance. It can enhance the chances of a hypoglycemic episode ten fold. I would recommend against it for any one not ABSOLUTLY comfortable with insulin or IGF1.
Here is how insulin and IGF1 work together. Igfbp3 is the binding protein, which allows IGF1 to remain active in the system for a long enough period of time to really work its magic. IGF1 by nature has a half-life of less than 10 minutes by its self. The molecule was so small it would escape the blood stream very rapidly. This was the reason IGF1 was so "underground". It took very frequent injections at high dosages to achieve even minimal results. Aside from this reconstituting the compound required a degree in biochemistry. This short acting version was the only IGF1 known until recently IGF1 would have been administered in 100 mcg dosages 4-6 times a day. That is a hell of a lot of IGF1. That explains a lot of the distended bellies. Now with R3 long chain IGF1 and the Binding protein IGFBP3 IGF1 will last up to 6 hours in the system. By binding IGF to the IGFBP3 you make the molecule larger and it gets trapped in the blood stream until the protein is broken down and the IGF molecule escapes. You can further its life by combining Insulin with it, although I here its very risky. Insulin prevents the breakdown of IGFBP3 and leaves the IGF1 molecule roaming free in the blood stream for longer periods of time up to 12 hours as insulin levels return to normal IGFBP3 will begin to break down and the IGF1 will escape from its bound protein IGFBP3 again having a half life of less than 10 minutes.
Insulin should be taken at the normal dosage it is usually administered at minus 10% about 45 minutes prior to the IGF1 infusion. Again let me remind you this can be deadly if you don't know what you are doing. And of course do not use Insulin for the nighttime injection of IGF1 by taking it in the morning you prolong the IGF1's half life to 12 hours and then take a 6 hour injection, you should be fine. Hell if you want to eat a big bowl of rice and drink another 100g of simple carbs 45 minutes before the bed time IGF1 infusion you could spike insulin for at least a few hours of extended IGF1 activity. If your not going to be using insulin in the stack then go ahead and do the same in the morning.
What users report
Users of IGF1 have reported various results but all along the same lines, It does not appear to be dramatically less effective in any one individual (at least not to the best of my knowledge). I have a good friend who had to stop taking IGF1 due to stomach illness that was completely unrelated But he to experienced good gains from it for the 2 weeks he was on it, his dosage was 120mcg per day. One hour after the first injection he went to the gym and immediately told me about the uncontrollable pump he got from just one set.
That would indicate to me that he was experiencing some form of cell volumization. The general consensus on IGF1 seems to be that its benefits are as fallow:
Increased Pump Pumps are reported to be so severe that workouts are often cut short due to lack of ability to the muscle through the full range of motion...ouch
Gains retention is increased if IGF is used in a cycle I am not sure why, but IGF1 seems to make gains on a cycle stick with virtually no post cycle loss. Every bodybuilder I've spoken with seems to think this for some reason. Most of them use drugs like Anadrol or Dianabol with it because of the amount of size attained with these drugs. The usual draw back to these drugs is that in most users there is a post cycle "crash" that occurs, so the reasoning is to toss IGF1 into the stack and grow larger faster with out the post cycle crash blues.
Reverses testicular atrophy
Testicles if shrunken will return to "full swing" so to speak even in the middle of a cycle. If not shrunken they will not shrink during the cycle. This may explain partially why gains are kept after the cycle.
Users report feeling drained and tired all day. This seems to be one of the negative side effects to IGF1, it will make you sleep longer and you will require more sleep at night to feel rested for the morning. This is common with high doses of HGH and exhibited in children, whose IGF1 levels are extraordinarily high. A child needs 4 hours more sleep than an adult on average does. This may be directly or indirectly related to IGF1 levels.
An almost arthritic feeling is commonly associated with high levels of HGH, well IGF1 has the exact same property. IGF1 will cause your hands, fingers and knuckles to ache this is one way you can be sure you got real IGF1.
IGF-1's Side effects
Every thing has a down side. To bake a cake ya gotta brake an egg. IGF1 is no exception. The drug used in larger quantity around the 100mcg+ range will cause headaches, occasional nausea and can contribute to low blood sugar or hypoglycemia in some users. Although I have never heard of this first hand I'm sure its true.
IGF1 will attach its self to the lining of the intestine and cause atrophy of the gut. Every thing IGF1 touches will grow and you have a lot of receptors on the lining of the large intestine and inner wall of the abdominal well. This is what causes the GH gut look. You can easily avoid this by limiting your dosages and cycle lengths. IGF1 cycles should be kept to 4-6 weeks with 4-6 weeks off in-between. IGF-1 is considerably more powerful than HGH and you need to think of it along those lines as far as dosing goes. We all know what to much HGH can do over prolonged periods of usage. The Neanderthal look is definitely not going to win any shows this year. I would recommend 80 mcg a day for 4 weeks at a time you should get good results from that for a while. I don't know if you will need to up the dosage at any point, but I would think in the case of IGF1 it wouldn't matter. If 80mcg doesn't do it for ya, then bump it up to 100 You should definitely feel it at this point If not suspect the IGF1 as being fake. Beyond 120 mcg per day your asking for trouble, This compound demands as much respect as its sister amino Insulin.
Clinical Facts about IGF-1
IGF-1 is a polypeptide of 70 amino acids (7650 daltons), and is one of a number of related insulin-like growth factors present in the circulation. The molecule shows approximately 50% sequence homology with proinsulin and has a number of biological activities similar to insulin. IGF-1 is a mediator of longitudinal growth in humans or how tall you are capable of becoming. Serum IGF-1 concentrations are altered by age, nutritional status, body composition, and growth hormone secretion. A single basal IGF-1 level is useful in the assessment of short stature in children and in nutritional support studies of acutely ill patients. For the diagnosis of acromegaly, a single IGF-1 concentration is more reliable than a random hGH measurement (Oppizi, et al., 1986). IGF-1 can be used for the assessment of disease activity in acromegaly (Barkan, et al., 198.
Almost all (>95%) of serum IGF-1 circulates bound to specific IGF binding proteins (IGFBPs), of which six classes (IGFBPs 1-6) have been identified (Rudd, 1991). BP3 is thought to be the major binding protein of IGF-1.
08-04-2004, 09:40 PM #4
Check out the big brain on DB LOL - nice info Bro - strangely familiar .....
08-04-2004, 09:50 PM #5
WHHOOOOOOHOOOOOOOO.....thanks brotha. Do some research and contact MR, thanks.
08-04-2004, 10:41 PM #6Banned
- Join Date
- Mar 2004
im 21 is that too young to use ifg-1, how much should it cost if i choose to do 100mcg a day....
08-04-2004, 10:49 PM #7Originally Posted by JoeyJuice
08-04-2004, 10:51 PM #8Banned
- Join Date
- Mar 2004
what about my age???how much does it cost
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