Long R3IGF-I

[nsa]

How much Long R3IGF-I would a first timer need to take and for how long to see results? Long R3IGF-I is different than regular IGF-1 right?

[bignatt]

Posted by JohnnyB


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.

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 (R), 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 (R) 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
centrifugation.

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.

[bignatt]

IGF FAQ
From Dr JMW@ AS

Long™R3IGF-I is an analog of human IGF-I.
· It is a superior alternative to insulin in serum-free media.
· It increases protein production by cells in culture medium.
· It increases cell viability by inhibiting apoptosis.
· It has a longer half-life in cell culture than insulin.
· It is readily available.
· There is secure and ample manufacturing capacity at GroPep Limited.
· No animal- or human- derived material is used in the manufacture or storage of Long™R3IGF-I.
· Long™R3IGF-I is already being used in the manufacture of three (3) biopharmaceuticals approved by FDA and EMEA.

Frequently Asked Questions

What cell types will respond to Long™R3IGF-I?
All cells that have a Type I IGF receptor will potentially respond. Most commercially used cells including CHO, fibroblasts and hybridomas have a type I IGF receptor. All cells which respond to pharmacological concentrations of insulin (>1 mg/liter) will respond to Long™R3IGF-I (10-50 mg/liter).

Is storage of the stock solution at 4°C acceptable?
Yes

How long is the stock solution stable for under these storage conditions?
Liquid stability data shows that Long™R3IGF-I is stable for 3 years (-20°C to 37°C). Therefore, the stock solution should be stable at 4°C for 3 years.

What type of preparation is available?
Liquid formulation, preferable for GMP production.
Freeze dried preparation.

Is Long™R3IGF-I stable?
Re-test date for freeze-dried peptide is 3 years. Liquid formulation stability studies have recently been completed. It is stable for 3 years (-20°C to +37°C). We have data indicating stability in media at 4°C for 1 year.

Here is an article written by a self-experimenter.

December 15, 2000

Answer: What a perfect question! You actually have talked to just the right person. I have a business associate that worked for the company that produces this in Australia. Several years ago, I ordered 10mg of Long R3 IGF-1 and used it for several months. What I found out was truly amazing.

Before I tell you about my results, let me tell you that if you are going to use IGF-1 then make sure it is the Long R3 version! Let me explain. Regular IGF-1 like what is produced in your body is transported around connected to binding proteins. There are quite a few of these and their main purpose is to grab ahold of the IGF-1 peptide and keep it from being quickly degraded. Without these binding proteins, all of the IGF-1 would be metabolized in the body within a few minutes. The problem (at least it seems like a problem but might actually be a good thing) is that these binding proteins basically prevent the IGF-1 from performing its function. As long as IGF-1 is attached to the binding protein it cannot do the cool stuff that it wants to do. Regular IGF-1 must be released from its binding protein in order to accomplish its mission. Part of the problem is that much of the IGF-1 is degraded before it is released (seems like much is wasted doesn’t it?)

With Long R3 IGF-1 this problem doesn’t exist. Understand that the Long R3 version does not bind to the various binding proteins. It is free to move throughout your body and immediately start doing all the cool stuff that it wants to do. Again, understand that the Long R3 version is several orders of magnitude stronger than regular IGF-1.

If you would happen to use regular IGF-1, you would need several milligrams per day in order to get the desired effect. With the Long R3 version, you need only microgram quantities. Long R3 is also inherently MUCH cheaper to produce. What I am saying is that for the average person, regular IGF-1 is not practical-it is too expensive and you need to use too much. With Long R3 IGF-1, the price to results ratio is pretty good!

Something else I want to explain is how I went about preparing it for injection into my body. Unfortunately, this is not easy and the average person will have a hard time doing it. At the time, I worked in a sophisticated lab which had all of the necessary equipment. I ordered 10mg of Long R3 IGF-1 and it came in a single flip-top vial. 10mg might not seem like much but believe me, when it comes to Long R3 IGF-1, it is a ton! Some people might say to just add saline to the vial, keep it in the fridge and inject it when necessary. However, this will not work well because the IGF-1 is not highly stable and will degrade in an aqueous environment. 10mg was enough for many months and I needed a way that would allow the IGF-1 to remain potent during this entire time. I did my research and developed my method. I ordered what is known as microvials and sterilized them. I then diluted the IGF-1 with sterile water and added just a tad of acid to increase stability. Although it took quite a while, I then used a micropipette and alliquotted an amount of solution that contained 50mcg into one of my microvials. I closed the microvial and then froze it in a deep freezer. When I was ready to inject, I took out one or more of my microvials, thawed it out, combined it with saline and injected it.

When I first started taking Long R3 IGF-1, I used 50mcg every other day. Amazingly, within days, I started noticing some effects in my body. I felt super hungry all of the time and just felt “anabolic ”. I can’t describe this feeling except to say that it was very similar to being on anabolic steroids (I wasn’t on at the time). Within one month, I gained almost 17 pounds of fairly lean mass! After the first month, something happened though and I noticed that it didn’t seem to be working that well. I upped the dosage several times over the next month to keep up the desired effects. On the third month, I was using several hundred micrograms per day but wasn’t noticing any further gains. All in all, I gained about 20 pounds of pretty solid mass!

Please notice that almost all of my gains were within the first month of taking the Long R3 IGF-1. After this first month, my gains slowed down considerably and eventually stopped altogether even though I was taking high dosages. Why did this happen?

From all of my research, I suppose one of two things might have happened to prevent me from making further gains. What I truly suspect is that the Long R3 IGF-1 downregulated the amount of binding proteins being produced by my body (research confirms this). When I first started to inject the IGF-1, I was supplementing my own body’s IGF-1. I not only had my own IGF-1 working throughout the day but I had the potent surges of Long R3 IGF-1 that I would inject. Over time though, the binding proteins were downregulated. Of course my body continued to produce some (albeit less) IGF-1, however, because there were very little or no binding proteins it was quickly degraded. From what I can tell, I was in a state where 95% of the day my body did not have the benefits of IGF-1. Basically, it got what it got when I injected the Long R3 version.

The other possibility is that I built up antibodies to the Long R3 IGF-1 which basically sought out and destroyed what I injected. Although possible, I don’t believe this actually happened because it is not supported by research. I have seen no evidence which suggests that Long R3 IGF-1 causes antibody production.

To fix the above problem, one would have to cycle the Long R3 IGF-1. The best thing would probably be to take it every other month. This would allow your own body’s IGF-1 and binding proteins to return to normal.

Overall, I had a good experience with Long R3 IGF-1. The results were different than with steroids . I have noticed that steroids cause preferential growth of certain muscles, especially those that are stressed (as in lifting). The IGF-1 though seemed to cause my entire body to get a little thicker. I guess IGF-1 is less compensatory in nature and exerts a more whole-body anabolicity.

Would I recommend IGF-1? To the right person who is very careful and knows what he’s doing and has a good background in the sciences and has access to a good lab, YES! However, you can tell that I have listed many prerequisites to using it. For the average Joe, I believe is is just too complicated to be safe.

[nsa]

Where your injections IM or subQ? SubQ is supposed to theoretically give better sustained levels than IM from what i understand, am i correct in this?

[nsa]

Thanks alot for your help with this topic. I don't think im going to use Long R3IGF-I anytime soon. I just got access to it for a pretty good price, so i wanted to research a little about it.