LR3 IGF-1

[JohnnyB]

LongR3 IGF-I
Good Science. Great Performance.


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 ®, 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
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.

JohnnyB

	IGF-1

	protein