Lysozyme as a Stability Study Model
Hen egg white lysozyme (HEWL, 14.3 kDa, EC 3.2.1.17) is the established model system for protein stability kinetics because its activity (lysis of Micrococcus lysodeikticus cell walls) is quantifiable by a validated turbidimetric assay (ISO 10272 adapted), its unfolding pathway is well characterised by DSC (Tm = 72°C at pH 7), and it degrades by a known combination of deamidation (Asn-Gly dipeptide motifs) and aggregation above pH 6.5. Our study used HEWL in three formulation matrices: (A) 50mM sodium citrate pH 6.0, (B) trehalose (10% w/v) + 50mM citrate pH 6.0, and (C) trehalose + mannitol (5%/5% w/v) + citrate pH 6.0 — representing progressively complex lyophilisation cryoprotectant matrices.
Accelerated & Real-Time Study Design
Following ICH Q1A(R2), real-time conditions (2–8°C, ongoing), intermediate (25°C/60%RH), and accelerated (40°C/75%RH) conditions were evaluated in sealed glass vials with rubber stoppers (residual O₂ <0.5%). Activity assays: turbidimetric assay at 450 nm, 0.15 mg/mL M. lysodeikticus suspension. Additionally, SDS-PAGE and SEC-HPLC (Agilent 1260, TSK-GEL G2000SWXL column) were used at each time point to characterise aggregation species. Points: T=0, 1, 3, 6, 9, 12, 18, 24 months for real-time; T=0, 1, 3, 6 months for accelerated.
Degradation Kinetics & Arrhenius Modelling
Activity loss followed first-order kinetics in all three matrices at all temperatures tested. Arrhenius plots (ln k vs. 1/T) were linear from 5°C to 40°C, yielding activation energies: Matrix A: Ea = 74.2 kJ/mol; Matrix B: 81.3 kJ/mol; Matrix C: 83.7 kJ/mol. The higher Ea in trehalose-containing matrices reflects preferential exclusion of trehalose from the protein hydration shell (Timasheff mechanism), slowing unfolding-dependent deamidation. At 40°C, Matrix A shows 12.4% activity loss at 6 months; Matrix C shows only 3.8% — a 3.3× stabilisation from the complex cryoprotectant blend. Predicted shelf life at 2–8°C (90% activity threshold): Matrix A = 31 months; Matrix C = 48+ months (extrapolated).
SEC-HPLC Aggregation Profile
SEC-HPLC at the 12-month real-time timepoint revealed: Matrix A: 2.3% high-MW aggregate species (>100 kDa), 4.1% low-MW fragments (<10 kDa). Matrix C: 0.6% aggregates, 1.2% fragments. All aggregate species in Matrix A were non-covalent dimers (confirmed by reducing SDS-PAGE). At 40°C/6 months, Matrix A: 18.4% aggregate; Matrix C: 4.7% aggregate. The aggregate threshold for IVD assay failure (>5% activity loss due to aggregation-mediated antigen bridging in immunoassays) is reached at 40°C/9 months for Matrix A, but not within the 6-month accelerated study window for Matrix C — consistent with the Arrhenius prediction.