Attention:

Certain features of Sigma-Aldrich.com will be down for maintenance the evening of Friday August 18th starting at 8:00 pm CDT until Saturday August 19th at 12:01 pm CDT.   Please note that you still have telephone and email access to our local offices. We apologize for any inconvenience.

LONG®R3IGF-I: An Insulin Alternative Exclusively for Cell Culture Applications

By: Jennifer Fries, BioFiles 2009, 4.5, 14.

LONG®R3IGF-I is a recombinant analog of human insulin-like growth factor-I (IGF-I) that has been specifically engineered for the enhancement of cell culture performance. LONG®R3IGF-I is more biologically potent in vitro than either insulin or native IGF-I and has been shown to significantly increase recombinant protein production. It is ideal for both research and large-scale culture systems utilizing serum-free or low-level serum applications.

LONG®R3IGF-I is specifically designed and manufactured exclusively for the research and industrial cell culture market for distribution by SAFC Biosciences.

back to top

LONG®R3IGF-I: Better science for better cell culture

Recombinant human insulin and other growth factors are essential for long-term growth and proliferation of cell lines. Although insulin is used as a growth factor in cell culture, its primary use is as a therapeutic drug for the treatment of diabetes. This has led to supply and availability issues for research and industrial cell culture users. In contrast, LONG®R3IGF-I is a dedicated raw material manufactured exclusively for cell culture applications providing a consistent, compliant and reliable alternative to recombinant insulin. It is not subject to market fluctuations and shortages as is recombinant insulin.

back to top

Proven science to maximize cell culture performance

When LONG®R3IGF-I is supplemented in serum-free medium it promotes cell proliferation, increased cell survival, increased productivity through greater proliferation and anti-apoptotic signaling. LONG®R3IGF-I provides equivalent or better performance to recombinant insulin depending on the cell line and clone. LONG®R3IGF-I has been used with numerous cell types including CHO, BHK, HEK 293, Vero, PER.C6®, MDCK and fibroblasts (graphs 1-4). All cell types that have a growth response to insulin in cell culture have the potential to respond to LONG®R3IGF-I.


Graph 1. CHO cell growth

Graph 2. CHO cell viability

Graph 3. CHO productivity

Graphs 1-3. Growth, viability and production data for insulin versus LONG®R3IGF-I. CHOK1 cells producing a recombinant protein were adapted to growth in protein-free media. Cells were cultured in spinner flasks in serum-free medium (60 mL) containing either insulin at 10 mg/L, LONG®R3IGF-I at 50 μg/L or no growth factor for a period of 11 days in a modified fed-batch process.

Graph 4. HEK 293 cell growth

Graph 4. Growth data for insulin, native IGF-I and LONG®R3IGF-I. HEK 293 cells were adapted to growth in a protein-free media. Cells were cultured in 250 mL shaker flasks in serum-free medium (60 mL) containing either native IGF-I at 100 μg/L, insulin at 1 mg/L, LONG®R3IGF-I at 100 μg/L or no growth factor for a period of 10 days in a batch process.

LONG®R3IGF-I is an analogue of human-like growth factor I (IGF-I) specifically engineered for use in research and industrial cell culture. Insulin, like IGF-I and their receptors (the insulin receptor (IR) and the type-I IGF receptor IGF-IR) have similar amino acid sequence and protein structure. As a consequence, insulin and IGF-I are able to bind to each other’s receptor with relatively low affinity. It is widely accepted that in CHO cells the effects of insulin are mediated by the IGF-IR, due to the fact there are relatively few IR present on the CHO cells and that insulin must be present at a high, non-physiological concentration typically 1–10 mg/L, to be effective. A more effective growth factor is one which targets and activates the IGF-IR directly, such as IGF-I or the analogue LONG®R3IGF-I. LONG®R3IGF-I has a distinct biological advantage over native IGF-I due to its low affinity for IGF Binding Proteins (IGFBPs). All mammalian cells secrete IGFBPs, which bind to and inhibit native IGF-I. The substitution of an arginine for glutamine acid at position three in LONG®R3IGF-I, in conjunction with the 13 amino acid N-terminal extension peptide, results in > 1000-fold reduced affinity for IGFBPs enhancing bioavailability and effectiveness in comparison to native IGF-I.

back to top

Increase cell density, maintain higher viability and extend

Under bioreactor conditions, stress induced apoptosis is the major cause of loss of cell viability. Activation of the IGF-IR results in the stimulation of a number of signal transduction cascades that have been identified as important for cell survival and proliferation. LONG®R3IGF-I not only results in greater activation of IGF-IR over insulin, but also results in greater activation of key anti-apoptotic and proliferative signaling molecules: Akt and MAPK. Increased activation of these signaling molecules prevents the loss of viability caused by different culture conditions.

back to top

Prolonged cell culture activity through greater stability

Under normal cell culture conditions, LONG®R3IGF-I is more stable than insulin, persisting up to two times longer. Insulin is degraded in cell culture by enzymes (insulinases) secreted by cells into culture media. In addition, insulin is more rapidly internalized and degraded compared with IGF-I and LONG®R3IGF-I. The extended cell culture stability of LONG®R3IGF-I results in prolonged activity and associated benefits to cell culture. (Graph 5).

 

Graph 5. LONG®R3IGF-I resulted in increased cell growth, viability and productivity (data not shown).

back to top

Regulatory compliant cGMP and animal free

LONG®R3IGF-I is manufactured under cGMP compliance with the International Conference on Harmonization (ICH) Q7A guidelines. The manufacturing plant is regularly audited by major European, US and Japanese biopharmaceutical companies. LONG®R3IGF-I is produced in a proprietary E. coli fermentation process that is completely animal free (AF). No animal-derived materials are used during the manufacture or storage of LONG®R3IGF-I. A certificate of origin is available upon request. Manufactured by Novozymes Biopharma, LONG®R3IGF-I has been supplied to the research and industrial cell culture market for over 15 years. In addition, LONG®R3IGF-I is used in the manufacture of several FDA (US), EMEA (Europe) and MHLW (Japan) approved marketed products, with many more in late-phase clinical trials.
 

Test Specification
Appearance Lyophilized white/creamy crystalline powder or a clear liquid
Endotoxin < 0.10 EU/μg protein
Bioburden Total viable aerobic count ≤100 cfu/mL
Biological Activity ED50 <10 ng/mL (Bioassay assessing the stimulation of protein synthesis in L6 myoblasts)
Concentration 0.9–1.1 mg/mL by reverse-phase HPLC
Identity Confirmed by N-terminal sequence analysis and reverse-phase HPLC (18 residues >95% single sequence)
Purity A single band ≥95% as determined by SDS-PAGE

 

LONG® is a registered trademark of Novozymes Biopharma AU. Covered by the following patents assigned to Novozymes Biopharma: US5,330,971 and 5,164,370; European patent 429,586, Australian patent 633,099 and Canadian patents 2,033,176 and 1,341,204.

PER.C6® is a registered trademark of Crucell N.V.
 

Product Description Source Recombinant Host Physical Form Assay Cell Type Affected Cat. No.
LONG®R3IGF-I human Escherichia coli lyophilized powder ≥95% SDS-PAGE - 85580C-1MG
85580C-10MG

 

Materials

     
Related Links