LL-37 — analytical characterisation
Chromatographic conditions, identity, related substances, presentation, reconstitution and in-use stability.
§6Analytical characterisation
§6.1Chromatographic conditions
- Column
- C18 or C8, 4.6 × 250 mm, 5 µm, 300 Å wide pore; a strongly cationic 37-mer requires an ion-pairing mobile phase for acceptable peak shape
- Mobile phase and gradient
- A: 0.1 % trifluoroacetic acid in water; B: acetonitrile with 0.1 % trifluoroacetic acid. Gradient 20–50 % B over 40 min at 40 °C
- Detection
- UV 214 nm; 258 nm (five Phe residues, no Trp or Tyr) — so 280 nm is again a poor choice
- Retention
- Intermediate to late; peak shape is the diagnostic feature, and a broad or tailing main peak indicates inadequate ion pairing or on-column aggregation
§6.2Identity by mass spectrometry
[M+5H]⁵⁺ at m/z ≈ 899.7, [M+6H]⁶⁺ at m/z ≈ 749.9. A highly basic peptide shifts the charge envelope to high charge states. Average mass 4493.3 ± 3 Da.[3]
§6.3Related substances and degradation
Table 7. Related substances recorded for LL-37, with the process or storage route that generates each and its analytical signature.
| Related substance | Origin | Analytical signature |
|---|---|---|
| Truncated chains | Incomplete coupling in a difficult 37-residue synthesis | Multiple species; a polycationic 37-mer has low crude purity and a 95 % final purity claim implies substantial purification |
| Deamidated Asn30 and Gln22 | Storage | +1 Da each |
| Aspartimide at Asp4 or Asp26 | Base-mediated side reaction | −18 Da |
| Aggregated species | On-column and in-solution self-association | Requires size-exclusion; an amphipathic cationic helix self-associates readily |
| Trifluoroacetate counter-ion | Purification | Six arginine and five lysine residues bind counter-ion stoichiometrically; trifluoroacetate content above 12 % by mass is expected and, critically, trifluoroacetate is itself cytotoxic to cultured cells at those concentrations, so residual counter-ion can confound bioactivity assays |
Degradation routes
- Deamidation
- Aspartimide formation
- Aggregation
- Proteolysis in any biological matrix
§7Presentation, reconstitution and storage
§7.1Presentation and reconstitution
- Presentation
- Lyophilised powder in vial; investigational topical and intralesional preparations
- Reconstitution
- A 1 mg vial with 1.0 mL gives 1 mg/mL. Cationic peptides adsorb strongly to glass and to polypropylene; low-binding tubes and a carrier such as 0.1 % bovine serum albumin or acetic acid are required to avoid substantial content loss at these concentrations.
- Storage, lyophilised
- −20 °C or below, desiccated
- Storage, reconstituted
- 2–8 °C for short periods; adsorptive loss is the dominant practical problem rather than chemical degradation
- In-use period
- No supported claim
For cationic antimicrobial peptides the counter-ion is not a bookkeeping detail. Trifluoroacetate at the levels typical of unexchanged material is cytotoxic to cultured cells, which means a bioactivity result obtained on trifluoroacetate-form peptide may reflect the counter-ion rather than the peptide. Acetate exchange is the standard remedy and should be documented.
§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.