IGF-1 LR3 — analytical characterisation
Chromatographic conditions, identity, related substances, presentation, reconstitution and in-use stability.
§6Analytical characterisation
§6.1Chromatographic conditions
- Column
- Size-exclusion chromatography for aggregates and correct folding; reverse-phase C4 or C8 wide-pore for intact analysis; peptide mapping for sequence confirmation
- Mobile phase and gradient
- Method-dependent
- Detection
- UV 280 nm
- Retention
- Not applicable in the small-peptide sense
§6.2Identity by mass spectrometry
Intact mass by high-resolution MS should return approximately 9111 Da. Three disulfide bonds must be verified as correctly paired, which requires peptide mapping under non-reducing conditions — an analysis no research-supply certificate assessed by the Institute has included.[3]
§6.3Related substances and degradation
Table 7. Related substances recorded for IGF-1 LR3, with the process or storage route that generates each and its analytical signature.
| Related substance | Origin | Analytical signature |
|---|---|---|
| Misfolded and scrambled-disulfide species | Refolding failure after bacterial expression | The dominant quality risk for any recombinant protein with three disulfides; biologically inactive or aberrantly active, and invisible to reverse-phase purity |
| Aggregates | Refolding and storage | Size-exclusion chromatography |
| Host-cell protein and endotoxin | Bacterial expression | Immunoassay and the bacterial endotoxin test. Endotoxin is a specific concern for any bacterially expressed protein and is rarely reported |
| Truncated and clipped species | Proteolysis during production | Reduced electrophoresis or peptide mapping |
| Residual methionine at the N-terminus | Incomplete processing | +131 Da |
Degradation routes
- Aggregation
- Disulfide scrambling
- Deamidation
- Oxidation
§7Presentation, reconstitution and storage
§7.1Presentation and reconstitution
- Presentation
- Lyophilised powder in vial (research supply only)
- Reconstitution
- Research vials are commonly 1 mg. A 1 mg vial with 1.0 mL gives 1 mg/mL, at which 50 µg is 0.05 mL, that is 5 units on a U-100 syringe.
- Storage, lyophilised
- −20 °C or below, desiccated
- Storage, reconstituted
- 2–8 °C for short periods; recombinant proteins in dilute solution without a carrier protein adsorb to container surfaces and lose content measurably
- In-use period
- No supported claim
The Institute includes this monograph principally to record a boundary: a bacterially expressed, three-disulfide recombinant protein cannot be assessed by a peptide purity certificate, and the assays that would matter — correct disulfide pairing, aggregate content, endotoxin — are not being performed anywhere in this supply channel.
§7.2In-use stability
Applicable standards: CEI-MS-01 · CEI-MS-02 · CEI-MS-03 · CEI-MS-04 · CEI-MS-05 · CEI-MS-06. The full series is at methodological standards.
Working calculators: reconstitution and insulin-unit conversion · purity against peptide content · certificate minimum-data checker.
References cited on this page
References are numbered in order of first citation in this document. Each superscript in the text links to its entry below.
- United States Pharmacopeial Convention. General Chapter ⟨1225⟩ Validation of Compendial Procedures. United States Pharmacopeia — National Formulary (USP–NF) 2024;USP 2024 Issue 1. identifier not held by the Institute
- International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use. ICH Q2(R2) Validation of Analytical Procedures. ICH Harmonised Guideline 2023;Step 4 version, 1 November 2023. identifier not held by the Institute
- International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use. ICH Q6B Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products. ICH Harmonised Tripartite Guideline 1999;Step 4 version. identifier not held by the Institute
- International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use. ICH Q1A(R2) Stability Testing of New Drug Substances and Products. ICH Harmonised Tripartite Guideline 2003;Step 4 version. identifier not held by the Institute
- Manning MC, Chou DK, Murphy BM, Payne RW, Katayama DS. Stability of protein pharmaceuticals: an update. Pharmaceutical Research 2010;27(4):544–575. doi:10.1007/s11095-009-0045-6 · PMID 20143256
Identifiers are reproduced only where the Institute holds them. Where a digital object identifier or PubMed identifier is not shown, the Institute has recorded the journal and year and has not constructed an identifier.