Frequently Asked Questions | LONG® R³ IGF-I 

No, LONG^® R3 IGF-I is a cGMP grade recombinant protein produced in E. coli. It is specifically manufactured for mammalian cell culture using a process that is free of animal-derived components. LONG^® R3 IGF-I is regulatory compliant and is currently used in the manufacture of several biopharmaceuticals approved by the FDA (United States), EMEA (Europe) and MHLW (Japan).

LONG^® R³ IGF-I is manufactured in a proprietary, but conventional, expression system. Cells taken from a validated working cell bank are fermented in a fed-batch process using fully defined animal component free media. Recombinant LONG^® R³ IGF-I is then isolated from inclusion bodies, refolded in redox buffer, and purified by a four-stage chromatography process.

LONG^® R³ IGF-I is available as a lyophilized powder, and a 1mg/mL liquid formulation in 100 mM acetic acid.

LONG^® R3 IGF-I is available from Repligen as a freeze-dried powder in 5 or 50 mg size vials. The 1 mg/mL liquid formulation of LONG^® R3 IGF-I is available in 5 mL, 100 mL, 250mL, 500mL and 1,000mL sizes.

Appearance, product identity (RP-HPLC, mass spectroscopy), purity, biological activity, bioburden and bacterial endotoxin analysis.

Lyophilized LONG^® R³ IGF-I must be reconstituted prior to use.

1. The product is supplied in an atmosphere of nitrogen at a slight vacuum (-25 kPa).
2. Remove the metal cap from the glass vial and introduce an air- filled syringe through the septum to equalize the pressure.
3. Add sufficient 100 mM acetic acid solution to the vial to achieve a concentration of 1 mg/mL LONG^® R³ IGF-I. We recommend keeping stock solution at ≥1 mg/mL.
4. Mix the solution thoroughly to ensure the peptide is completely dissolved.
5. Re-suspended LONG^® R³ IGF-I, or media containing LONG^® R³ IGF-I, may be filtered through a low protein binding membrane such as Polyvinylidene Difluoride (PVDF) or Polyethersulfone (PES) with a pore size of 0.22 μm.

 Liquid LONG^® R³ IGF-I is ready to use, there is no need to defrost or reconstitute. Simply open and dilute directly into cell culture media.

Studies have shown that a small percentage of LONG^® R³ IGF-I is lost during filtration. The following table shows the amount of LONG^® R³ IGF-I lost on specific types of filter membranes.

Yes, an Enzyme-Linked Immunosorbent Assay (ELISA) kit is available from Repligen for determining LONG^® R³ IGF-I concentrations in samples.

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We do not recommend that the powder be dissolved in cell culture media.

Like insulin and all small peptides LONG^® R³ IGF-I can non- specifically adsorb to plastic, glass and stainless steel surfaces in low protein-containing media. Often this adsorption is minimal and does not affect overall cell culture performance. However, incorrect and inconsistent sample handling procedures can impact the accuracy of LONG^® R³ IGF-I detection.

To minimize non-specific adsorption of LONG^® R³ IGF-I

• Reconstitute stock solutions of LONG^® R³ IGF-I at 1 mg/mL or greater.
• Always use filters with low protein-binding properties such as PVDF or PES.
• Where applicable, add LONG^® R³ IGF-I as far down the media manufacturing process as possible, preferably to the fermentation tank directly.

Lyophilized LONG^® R³ IGF-I is stable for five years when properly stored at 2 to 8^οC. Liquid LONG^® R³ IGF-I, in the original unopened vial, is stable for five years when stored at 2 to 8^οC.

Reconstituted lyophilized LONG^® R³ IGF-I, and open containers of liquid LONG^® R³ IGF-I, should be stored re-capped in the original vial at 2 to 8^οC.

Once reconstituted, the solution can be aliquoted into smaller sizes (i.e. for single use) in and stored in LoBind eppendorf tubes at 2-8°C. LoBind tubes are recommended to prevent protein adsorption. Solutions stored under such conditions can be used for 12 months.

To ensure consistency and accuracy in the measurement of LONG^® R³ IGF-I, we recommend the following:

• Standardize the procedures for sampling and handling of cell culture media samples.
• Determine the optimal low protein-binding tube type for handling and storage of samples.
• Avoid sub-sampling and minimize repeated exposure to surfaces.
• The presence of a carrier protein in the media or tube can offer protection against non-specific adsorption.
• Ensure all samples have equilibrated to room temperature prior to analysis.

Yes, an Enzyme-Linked Immunosorbent Assay (ELISA) kit is available from Repligen for determining LONG^® R³ IGF-I concentrations in samples.

Learn more

LONG^® R³ IGF-I has been shown to increase cell growth and overall volumetric productivity in CHO cell culture. In addition, LONG^® R³ IGF-I may enhance protein production by reducing apoptosis and extending culture duration.

All cells that have a Type I IGF receptor, or are insulin sensitive, will potentially respond to LONG^® R³ IGF-I. This group includes but is not limited to most commercially used cell lines such as Chinese Hamster Ovary cells (CHO), fibroblasts, hybridomas, embryonic stem cells (ES), natural killer cells (NK), mesenchymal stem cells (MSC), and hematopoietic stem cells (HSC).

Because of the large dilution of sterile LONG^® R³ IGF-I into cell culture media, there should be no effect on pH or osmolality.

The working concentration range for LONG^® R³ IGF-I is 10 - 100 μg/L. We recommend starting with 50 μg/L then  performing titrations to optimize concentrations with your cell line, process, and application.

Direct or sequential adaptation are methodologies that can be used for weaning cells into LONG^® R³ IGF-I containing media. Cells should be in mid-logarithmic growth phase and  ≥90% viability before starting the adaptation process.

1. Direct
   Some clones will not require weaning and can be grown immediately in media containing an appropriate quantity of LONG^® R³ IGF-I (typically 10 - 100 μg/L).
    
2. Sequential
   Less robust clones may need to be adapted to new media sequentially. Start by sub-culturing cells in a 25%:75% mixture of media with and without LONG® R³ IGF-I. When cell viabilities exceed 90%, and cell doubling times are stable, cells can be transitioned to the next media mixture (i.e. 50%:50%). If growth slows, or viability drops, cells should continue to be passaged in the same media until viability and doubling times stabilize

A combination of LONG^® R³ IGF-I and insulin may be beneficial for some cell lines. We recommend the amount of LONG^® R³ IGF-I be optimized for use with different cell lines, applications, and processes.

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