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. 产品保存在轻度真空(-25 kPa)的氮气环境中。
2. 从玻璃瓶上取下金属瓶盖，通过隔片连接一个充满空气的注射器，以平衡压力。
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. 充分混合溶液，确保肽完全溶解。
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.

Learn more

我们不推荐将粉末溶解在细胞培养基中。

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.
• 使用低蛋白质结合的过滤器，如PVDF或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.

复溶后，溶液可以分装成更小的规格（针对单次使用）并置于LoBind eppendorf小管内，于2-8°C储存。推荐使用LoBind管，以防止蛋白质吸附。在此条件下储存的溶液可使用12个月。

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

• 建立标准的细胞培养基样品取样和操作程序。
• 确定用于处理和储存样品的最佳低蛋白结合管类型。
• 避免二级取样，以降低重复暴露于表面。
• 培养基或管中存在载体蛋白时，可提供保护作用，降低非特异性吸附。
• 分析前，确保所有样品已平衡至室温。

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. 直接
   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. 顺序
   某些较不稳健的细胞可能需要逐步适应新的培养基。比如开始培养时，可以使用含和不含LONG® R³ IGF-I的培养基比例为25%:75%的混合液。当细胞活性>90%，且细胞倍增时间稳定时，将培养基置换为新的混合液（如 50%:50%）。如生长变缓或活性下降，细胞应在相同的培养基中继续传代，直到活性和倍增时间稳定。

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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