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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
???g/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 ???g/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 ???g/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 ???g/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 ???g/L) in cell culture medium containing the
recommended levels of LongR3 IGF-I can result in beneficial synergistic effects
in certain applications.
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