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.

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.

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

1. 生成物は、わずかな減圧（-25 kPa）の窒素雰囲気で供給されます。
2. ガラスのバイアルから金属キャップを取り外し、セプタムを通して空気を満たしたシリンジを導入して圧力を均一にします。
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. 溶液をよく混ぜて、ペプチドを完全に溶解させます。
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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細胞培養培地中に粉末を溶解することはお勧めしません。

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.
• タンパク質結合性の低いフィルター（PVDF、PESなど）を常に使用してください。
• 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.

元に戻した場合は、少量の溶液（シングルユース用）をLoBindエッペンドルフチューブに分注して、2～8℃に保持することで保管できます。タンパク質の吸収を抑えるために、LoBindチューブの使用を推奨します。このようにして保管された溶液は12か月間使用できます。

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

• 細胞培養培地サンプルのサンプリングと取り扱いの手順を標準化してください。
• サンプルの取り扱いや保管に最適な、タンパク結合率の低いチューブを使用してください。
• サブサンプリングを避け、表面への繰り返しの露出を最小限に抑えてください。
• 培地やチューブにキャリアタンパク質が存在すると、非特異的吸着が抑制されます。
• 分析前に、すべてのサンプルの温度が室温に保たれていることを確認してください。

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. シーケンシャル
   堅牢性の低いクローンの場合、新しい培地に段階的に適応させる必要があることがあります。LONG®R³ IGF-Iを含む培地と含まない培地を25%:75%の割合で混合した培地で、細胞を継代培養することから開始してください。細胞生存率が90％を超え、かつ細胞倍加時間が安定している場合、細胞を次の混合培地（50%:50%）に移します。成長が遅くなるか生存率が低下した場合、生存率と倍加時間が安定するまで、細胞を同じ培地で継代し続ける必要があります。

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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