1.0 Compound Identification
| Common Name | Semaglutide |
|---|---|
| CAS Number | 910463-68-2 |
| Molecular Weight | 4113.58 g/mol (average mass) |
| Class | Modified glucagon-like peptide-1 (GLP-1) analogue; 31-residue peptide with two non-natural amino acid substitutions (Aib8, Arg34) and a C18 fatty diacid side-chain at Lys26 via a two-unit mini-PEG linker |
| Structural feature | N-ε-{2-[2-(2-{2-[2-(2-{(4S)-4-carboxy-4-[10-(4-carboxybutanoylamino)decanoylamino]butanoylamino}ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl}-[Aib8,Arg34]GLP-1-(7–37) peptide |
| Appearance (lyophilized) | White to off-white powder |
| Purity specification | ≥98% by RP-HPLC (UV 220 nm, area percent) |
2.0 Reconstitution Protocol
The following protocol describes reconstitution of lyophilized semaglutide for laboratory analytical use. Reconstitution volumes assume a nominal fill of 5 mg per vial.
| Diluent | Bacteriostatic water for injection (0.9% benzyl alcohol) for multi-use applications; sterile water for injection for single-use aliquots |
|---|---|
| Volume | 1.0 mL per 5 mg vial (yields nominal 5 mg/mL stock solution) |
| Mixing method | Inject diluent slowly against the side of the vial wall; allow powder to dissolve; gently swirl for 20–30 seconds. Do not vortex. Do not shake. |
| Appearance after reconstitution | Clear, colorless to faintly yellow solution, free of visible particulates. Discard if turbid or contains visible particles. |
| Filtration | Optional: pass through 0.22 µm PVDF syringe filter before aliquoting for extended storage |
3.0 Stability Data
The table below presents HPLC-UV purity measurements (% of main peak, area percent at 220 nm) for reconstituted semaglutide solutions (5 mg/mL in bacteriostatic water) stored under three temperature conditions. Data are derived from internal stability studies using the RP-HPLC method described in Nexphoria Method Note NX-MN-003. Time 0 is defined as the date of reconstitution.
| Storage Condition | Day 0 (%) | Day 7 (%) | Day 14 (%) | Day 21 (%) | Day 28 (%) |
|---|---|---|---|---|---|
| 2–8°C, protected from light | 100.0 | 98.5 | 97.1 | 95.8 | 94.3 |
| 25°C, protected from light | 100.0 | 96.2 | 92.4 | 88.7 | 84.9 |
| 40°C, ambient humidity | 100.0 | 91.3 | 82.6 | 74.1 | 66.5 |
Results indicate that reconstituted semaglutide stored at 2–8°C maintains ≥94% purity through 28 days. Storage at 25°C or above results in accelerated degradation; solutions stored at 25°C fall below 90% purity between Days 14 and 21. Storage at 40°C is provided for reference under ICH Q1A accelerated conditions; solutions stored at 40°C are not intended for extended laboratory use [1, 2].
4.0 pH Stability Range
Semaglutide demonstrates acceptable chemical stability across a pH range of 6.5–8.0 when held at 2–8°C. Outside this range, increased degradation rates are observed: acidic conditions (pH <6.0) promote aspartate isomerization and hydrolytic cleavage at susceptible residues, while strongly alkaline conditions (pH >9.0) accelerate deamidation and peptide backbone hydrolysis [3].
| pH Range | Stability Assessment | Notes |
|---|---|---|
| 6.5–8.0 | Acceptable (≥94% retained at 28 d, 2–8°C) | Recommended operating range |
| 5.0–6.5 | Marginal | Accelerated Asp isomerization; avoid where possible |
| <5.0 or >9.0 | Not recommended | Rapid degradation observed; use only for short-term analytical manipulation |
5.0 Light Sensitivity
Semaglutide solutions exhibit photodegradation when exposed to direct UV or intense visible light. Tryptophan and phenylalanine residues within the GLP-1 backbone are susceptible to photo-oxidation. All reconstituted solutions should be stored in amber vials or protected from direct light. Laboratory handling should be performed under subdued lighting; direct exposure to fluorescent or UV lamp sources should be avoided during preparation and aliquoting [4].
6.0 Freeze-Thaw Cycle Tolerance
Reconstituted semaglutide solutions tolerate a maximum of three freeze-thaw cycles when stored at −20°C between cycles. Beyond three cycles, cumulative purity loss attributable to aggregation and degradation exceeds the 5% threshold based on internal data [5]. To minimize the number of freeze-thaw cycles, working solutions should be aliquoted into single-use volumes before initial freezing.
| Freeze-Thaw Cycle | Purity Remaining (%) | Assessment |
|---|---|---|
| 0 (reconstituted, no freeze) | 100.0 | Baseline |
| 1 | 99.1 | Pass |
| 2 | 97.8 | Pass |
| 3 | 96.2 | Pass (marginal) |
| 4 | 93.7 | Borderline; not recommended |
| 5 | 90.5 | Fail (<94% threshold) |
7.0 References
- [1] ICH Harmonised Guideline Q1A(R2): Stability Testing of New Drug Substances and Products. (2003). International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use. https://www.ich.org/page/quality-guidelines
- [2] Manning M.C., Chou D.K., Murphy B.M., Payne R.W., Katayama D.S. (2010). Stability of protein pharmaceuticals: an update. Pharmaceutical Research, 27(4):544–575. DOI:10.1007/s11095-009-0045-6
- [3] Capelle M.A., Gurny R., Arvinte T. (2007). High throughput screening of protein formulation stability: practical considerations. European Journal of Pharmaceutics and Biopharmaceutics, 65(2):131–148. DOI:10.1016/j.ejpb.2006.09.009
- [4] Kerwin B.A., Remmele R.L. Jr. (2007). Protect from light: photodegradation and protein biologics. Journal of Pharmaceutical Sciences, 96(6):1468–1479. DOI:10.1002/jps.20815
- [5] Jiang G., Ni N., Bhatt H., Han Q., Szymanski J., Bhatt D. (2009). Freeze-thaw cycling of proteins: effects on aggregation and activity. PDA Journal of Pharmaceutical Science and Technology, 63(5):359–369. PMID:19921979
- [6] United States Pharmacopeia (2024). USP <659> Packaging and Storage Requirements; USP <1151> Pharmaceutical Dosage Forms. In USP–NF. United States Pharmacopeial Convention.