Stability Assessment of hospital-produced anti-staphylococcal phage therapeutic medicinal Products

Context
Bacteriophages, natural viruses of bacteria, offer a potential therapy for difficult-to-treat infections. Developing Phage Therapeutic Medicinal Products (PTMP) requires long-term stability studies per ICH guidelines, complemented by Advanced Kinetic Modeling (AKM) to predict and mitigate instability risks early in highly variable biologicals. This study aimed to evaluate the stability of four hospital-produced anti-staphylococcal PTMP (Silviaviruses V1SA19, V1SA20, V1SA22; Rosenblumvirus V4SA02) using both AKM and ICH methodologies, comparing their outcomes.

Material and Methods
Purified PTMP batches (N=12) were formulated in the patented multi-component excipient solution PhageProtect, filtered (0.22 µm), and aseptically filled into type 1 glass vials (1.2 mL). Critical quality attributes (CQAs), in accordance to the Ph.Eur. 5.31. PTMP, included : potency (≥10⁸ PFU/mL, spot test), particle aggregation (mean diameter: 140±40 nm, interferometry), purity (>99.9%, qPCR), osmolality (400±40 mOsm/kg), pH (7.5±0.5), sterility, endotoxins (<350 IU/mL), particulate contamination (<6,000 particles/vial ≥10 µm; <600 ≥25 µm), and excipient concentration (sodium, potassium, phosphate, polysorbate 80, ±10% acceptance). AKM for V1SA19 was conducted at 5°C, 25°C, 30°C, and 40°C over 3 months, with analyses at D0, D7, D14, D28, D60, and D90 (N=3). Potency was modeled for 1.5 years using AKM, while the ICH study assessed all CQAs at 5°C±3°C and 25°C±2°C over 2 years (D0, M0.5, M1, M1.5, M2, M3, M6, M12, M18).

Results
AKM-predicted phage titers at 5°C were (8.8±0.2)E8 PFU/mL vs (6.9±3.0)E8 PFU/mL (ICH) at 12 months, and (8.2±0.3)E8 vs (5.5±2.7)E8 PFU/mL at 18 months. ICH results showed potency reductions of 0.6 log (V1SA19) to 1.8 log (V1SA22) PFU/mL at M18, with V4SA02 (initial 2×E8 PFU/mL) losing 1.6 log over 18 months. Mean particle diameters at 5°C remained stable (143±2 nm vs 143±7 nm, p=0.92), but V1SA20 at 25°C (M1) reached 239 nm, indicating temperature-dependent aggregation trend. Polysorbate 80 concentration was -28% at D0 and -32% at M18, though it remained above the critical micelle concentration (0.016 mg/mL at 25°C). All other CQAs met specifications.

Conclusion
This study demonstrates comprehensive PTMP stability over 2 years at 5°C and 25°C via ICH, with V1SA19 also evaluated via AKM. AKM provides early insights for candidate selection, crucial for these variable products. ICH identified two PTMP stable for ≥18 months, enabling practical use, while the other two require targeted production. Polysorbate 80’s sub-specification concentration prompted a reassessment of analytical methods then dilution process, but its active presence validated the stability data. Next, our center will study anti-Escherichia coli PTMP stability.