Module 3.2.S — Drug Substance (Full) — GLPI-103

[MOCK — deep-knowledge assumption] Full drug-substance section (S.1–S.7) for the GLPI-103 active pharmaceutical ingredient. Numerical values (sequence, mass, specifications, batch and stability data) are illustrative but technically representative of a lipidated synthetic-peptide GLP-1/APJ dual agonist.

Change History

S.1 General Information

• S.1.1 Nomenclature: GLPI-103 (development code); INN proposed; CAS (to be assigned). Chemical class: synthetic, lipidated, linear analogue peptide acting as a dual agonist of the human GLP-1 receptor (GLP-1R) and the apelin receptor (APJ).
• S.1.2 Structure: a 39-amino-acid single-chain peptide bearing (i) α-aminoisobutyric acid (Aib) at positions 2 and 13 to confer dipeptidyl-peptidase-4 (DPP-4) and general protease resistance, (ii) a C18 fatty di-acid (octadecanedioic acid) albumin-binding moiety conjugated to the Lys²⁰ ε-amine through a γ-Glu + 2×AEEA (8-amino-3,6-dioxaoctanoic acid) spacer to extend plasma half-life, and (iii) a C-terminal amidation. The APJ pharmacophore is engineered into the C-terminal domain. Molecular formula ≈ C₂₂₁H₃₄₈N₅₄O₆₈ ; average molecular mass ≈ 4,914 Da (monoisotopic ≈ 4,911 Da). One intramolecular lactam bridge stabilises the helical segment.
• S.1.3 General properties: white to off-white lyophilised powder; freely soluble in water and aqueous buffers at pH ≥ 7.0, sparingly soluble at the isoelectric point (pI ≈ 4.7); hygroscopic; specific optical rotation and UV (λmax ≈ 280 nm, Trp/Tyr) characteristic; supplied as the acetate salt.

S.2 Manufacture

• S.2.1 Manufacturer(s): named GMP manufacturing site(s) operating under ICH Q7.
• S.2.2 Description of manufacturing process and process controls (Q11):
  1. Solid-phase peptide synthesis (Fmoc/tBu) on a low-loading resin, stepwise coupling with DIC/Oxyma activation; selected difficult couplings performed as pre-formed dipeptide fragments to control deletion sequences.
  2. On-resin side-chain conjugation of the γ-Glu–2×AEEA–C18-diacid lipid to the Lys²⁰ side chain (orthogonal ivDde deprotection).
  3. Global cleavage/deprotection (TFA/scavenger cocktail) and ether precipitation of crude peptide.
  4. Preparative reversed-phase HPLC purification (C18, acetonitrile/aqueous TFA then acetate salt exchange) and lyophilisation.
  • Critical process parameters / in-process controls (IPCs): coupling completion (Kaiser/chloranil, ninhydrin ≤ 0.5% free amine); crude purity by RP-HPLC; main-pool purity ≥ 95.0% with single max impurity ≤ 1.0% before lyophilisation; residual TFA after salt exchange.
• S.2.3 Control of materials: Fmoc-amino acids, resin, reagents and solvents controlled to compendial/internal specifications; TSE/BSE-free declarations for any animal-derived material (none used).
• S.2.4 Controls of critical steps and intermediates: defined acceptance criteria for the crude peptide and the purified intermediate.
• S.2.5 Process validation/evaluation: prospective validation at registration scale (≥ 3 consecutive batches) — see M3-PV.
• S.2.6 Manufacturing development history: scale-up from discovery (solution-assisted) to commercial SPPS; comparability assessed across changes per ICH Q5E principles.

S.3 Characterisation

• S.3.1 Elucidation of structure: primary structure confirmed by high-resolution LC-MS (intact mass and reduced), tryptic/Glu-C peptide mapping with MS/MS, total amino-acid analysis, N-terminal Edman sequencing and C-terminal characterisation; site of lipidation confirmed by MS/MS; higher-order structure by circular dichroism (α-helical content) and analytical SEC.
• S.3.2 Impurities: process-related — single amino-acid deletion (des-Gly, des-Ser) and insertion sequences, D-/epimerised residues, incomplete-acylation (des-lipid) species, oxidation (Met/Trp), deamidation (Asn→isoAsp), acetylation/trifluoroacetylation, and residual reagents/solvents; product-related — dimers/aggregates and the diketopiperazine N-terminal truncation. Each impurity ≥ identification threshold is structurally characterised and toxicologically qualified (Q3A).

S.4 Control of Drug Substance

S.4.1 Specification (illustrative)

S.4.2–S.4.3 Analytical procedures & validation

Procedures validated per ICH Q2(R2) (specificity incl. forced-degradation peak purity, accuracy, repeatability/intermediate precision, linearity, range, LOD/LOQ, solution stability, robustness); details in M3-AMV.

S.4.4 Batch analyses

S.4.5 Justification of specification

Limits are justified by manufacturing capability (batch data), the stability-study degradation profile, toxicological qualification of individual and total impurities (Q3A), potency–efficacy linkage, and compendial requirements; the assay and related-substance methods are stability-indicating (forced degradation: acid/base hydrolysis, oxidation H₂O₂, thermal, photolytic, all with peak-purity confirmation).

Impurity investigation (QA-003 ISS-011): an early development lot showed a des-Gly deletion-sequence impurity at 0.18% (above the identification threshold). Root cause = incomplete coupling at a sterically hindered residue. The lot was re-purified and the impurity was structurally characterised and toxicologically qualified (Q3A); CAPA changed the difficult coupling to a pre-formed dipeptide fragment and added a coupling-completion IPC (M3-CS §2), which controls the impurity to ≤0.10% in subsequent batches.

S.5 Reference Standards or Materials

A fully characterised primary reference standard (orthogonal mass, sequence, potency, water/counter-ion) and a qualified working standard bracketed against it; requalification on a defined interval and on each new lot.

S.6 Container Closure System

Drug substance stored in double low-density-polyethylene (LDPE) liners within an HDPE drum, with desiccant, protected from light; extractable/leachable and moisture-protection suitability demonstrated.

S.7 Stability (Q1A(R2)/Q5C)

Stability-indicating RP-HPLC (assay, related substances) and SE-HPLC (aggregates) on registration batches:

Principal degradation pathways are oxidation (Met/Trp), deamidation (Asn), and aggregation, all controlled by the specification and the recommended −20 °C storage with a 24-month re-test period (full dataset in M3-STAB).