IGF-1 LR3 Stability: How It Degrades and How to Slow It

IGF-1 LR3 is less forgiving in solution than the short peptides in this series because it is a folded protein. Its methionines oxidise with light, oxygen and warmth; its three disulfide bridges can rearrange; it can aggregate when shaken or frozen; and when dilute it is lost to surfaces. Supplier figures for the refrigerated solution run from one week to one month, so a mixed vial is best used early.

Reading the structure

IGF-1 LR3 is an 83-residue chain: a 13-residue extension, MFPAMPLSSLFVN, joined to human IGF-I with arginine at IGF-I position 3 (OriGene, AR31185PU-S). Against the usual degradation routes in proteins (Manning et al., Pharmaceutical Research, 2010):

• Three methionines. One matches methionine 59 of IGF-I.
• Six cysteines in three disulfide bridges. They hold the fold, and they can rearrange.
• An Asn-Gly bond. The extension’s last residue, asparagine, is followed by IGF-I’s first residue, glycine. Asn-Gly is one of the fastest-deamidating sequences in model peptides.
• No tryptophan or histidine, and no Asp-Gly or Asp-Pro bonds.
• A hydrophobic extension. It helps the chain fold (Francis et al., Journal of Molecular Endocrinology, 1992), and like any folded protein the molecule can unfold and clump at surfaces.

Methionine oxidation

Formulation studies on human IGF-I followed the oxidation of its methionine 59. The rate depended on the amount of protein and of dissolved oxygen, and light exposure, storage temperature and oxygen content all had a considerable effect; there was no significant difference between solution and the solid state (Fransson et al., Pharmaceutical Research, 1996). Visible light and phosphate buffer both increased it (Fransson and Hagman, Pharmaceutical Research, 1996), and a later study found it catalysed by light and by ferric ions combined with EDTA (Fransson, Journal of Pharmaceutical Sciences, 1997). The solid-state finding is why the dry powder is also kept dark and sealed.

Disulfide scrambling and dimers

IGF-I folds into two disulfide arrangements of similar stability: the native pairing and a “swap” form in which two bridges trade partners. Without a free thiol each form is stable indefinitely at neutral pH, but with one they interconvert (Miller et al., Biochemistry, 1993). One of the three bridges is strained (Hober et al., Biochemistry, 1992). In the formulation work, light and ferric ions drove covalent dimers, formed both by disulfide scrambling and by a radical pathway (Fransson, 1997). For a vial: no reducing agents, no light, clean water.

The Asn-Gly junction

In model peptides an Asn-Gly bond deamidated with a half-life of only 1.4 days at 37 °C and pH 7.4 (Geiger and Clarke, Journal of Biological Chemistry, 1987). When making IGF-I from a longer fusion protein, the same group used a Val-Asn-Gly link precisely because the Asn-Gly bond can be cut chemically, with hydroxylamine (King et al., Journal of Molecular Endocrinology, 1992). In IGF-1 LR3 that bond stays in place, joining the extension to IGF-I. What deamidation there does to activity has not been measured that we could find. Cold slows it.

Aggregation and surfaces

Shaking creates air and liquid interfaces where folded proteins unfold and clump, and freezing concentrates protein at ice boundaries (Manning et al., 2010). At low concentration the problem reverses: a larger share of the protein sticks to glass and plastic. The MedChemExpress sheet does not recommend reconstituting below 0.1 mg/mL and advises adding a carrier protein such as 0.1% albumin for long-term storage of solution (MedChemExpress, HY-P70783). A 1 mg vial mixed with 1 to 5 mL sits at 1 to 0.2 mg/mL, inside the range OriGene gives for reconstitution.

Lyophilised versus reconstituted

MedChemExpress rates the lyophilised protein at 2 years at minus 20 °C. OriGene rates it at 6 months at 2 to 8 °C or long term at minus 20 °C, with a shelf life of one year from despatch. Days at room temperature in transit do not harm sealed powder.

Once mixed, the supplier figures are short. MedChemExpress gives reconstituted protein 1 week at 4 °C; OriGene gives the undiluted solution one month at 2 to 8 °C and diluted solutions 1 week. Those are for plain water without a preservative, not bacteriostatic water, but they are the only published numbers. Our general handling window is 8 to 12 weeks at 2 to 8 °C in the dark, fading from around week 6 to 8, and for IGF-1 LR3 the sensible reading is to mix close to use and treat the early weeks as the best period. The full table is on how long IGF-1 LR3 lasts.

Temperature and light

The methionine oxidation in IGF-I had an activation energy of about 95 kJ/mol (Fransson et al., 1996), so warmth speeds it markedly. Light drove both oxidation and covalent dimers in every study above. A mixed vial goes back into its box in the main body of the fridge after each access.

Freeze-thaw

Sealed powder can be frozen. A mixed vial that will be opened again is not. Both supplier sheets recommend single-use aliquots at minus 20 °C or minus 80 °C for longer storage, and OriGene warns against repeated freezing and thawing.

What visible changes mean

Mixed IGF-1 LR3 is clear and colourless. Oxidation, deamidation and disulfide scrambling are invisible, so a clear vial is not on its own proof of full strength. Discard the solution if it shows:

• Cloudiness that does not clear within 10 to 15 minutes of mixing, or that appears later.
• Strands, flakes, particles or a gel.
• Any yellow or other tint.
• A history of being shaken hard, frozen and thawed, left out overnight or left in the light.

The general causes of haze and gel are covered in why peptides gel or go cloudy. Bench steps that protect stability are on the IGF-1 LR3 handling page.