Pharmaceutical Grade Sodium Hyaluronate

Host & Strain Strategy

• Preferred hosts (modern): Bacillus subtilis (GRAS), Corynebacterium glutamicum.
• Legacy host: Streptococcus zooepidemicus (high titers; higher ET/pyrogen risk).
• Engineering targets: Overexpress hasA/B/C (HA synthase, UDP-GlcUA, UDP-GlcNAc pathways), boost ugd, glmS/glmU, reduce hyaluronidase, tune capsule/export genes.
• Select for: High MW stability (low hyaluronidase), low lysis, robust at pH ~7.

Upstream: Media & Seed

Chemically defined or semi-defined to lower impurities/endotoxin (ET).

• Carbon: Glucose or sucrose (start 30–40 g/L; fed-batch 2–5 g/L residual).
• Nitrogen: Low-ET yeast extract/peptone or defined amino nitrogen.
• Salts/trace: MgSO₄ (0.5 g/L), K₂HPO₄/KH₂PO₄ (1–2 g/L).
• Seed train (typical): 50 mL (250 mL flask) → 0.5–1 L (2 L flask) → 2–3 L seed STR, all 35–37 °C, pH ~7, 6–10 h per stage, inoculate production at 5–10% v/v (late-exp phase).

Fermentation (batch→fed-batch)

• Scale: 2–5 L (dev), 10–50 L (pilot); Temp: 35–37 °C; pH: 6.8–7.2
• DO: ≥30% (prefer O₂ enrichment before raising rpm)
• Agitation: Low-shear impellers; tip speed ~1.5–2.0 m/s; Aeration: 0.5–1.0 vvm
• Antifoam: Pharma-grade silicone/PPG (track carryover)
• Mode: Start batch; feed glucose to hold 2–5 g/L residual
• Harvest: 12–18 h, when viscosity plateaus; HA 5–10 g/L typical

Notes: HA broth becomes highly viscous → manage shear carefully (shear reduces MW). Maintain pH/DO tightly; avoid glucose >10 g/L (overflow/depolymerization).

Primary Recovery & Clarification

• (Optional) Thermal kill 60–65 °C, 20–30 min (balance vs MW loss).
• Debris removal: Disc-stack or low-shear depth filtration train (GF → 1–3 µm → 0.45 µm PES/CA).
• Enzymatic clean-up (optional)
• Keep everything cool (15–25 °C) and low-shear.

Capture (Alcohol Precipitation)

• Cool filtrate to 4–10 °C; add 2–3 vol cold ethanol or IPA with gentle mixing.
• Hold 1–2 h; collect fibrous HA by basket centrifuge/Nutsche.
• Wash cake with 70–80% alcohol to remove salts/proteins/pigments.
• Dehydrate to a friable cake.

Notes: Avoid CTAB complexation for pharma APIs due to residual risk.

Redissolution & Polishing

• Redissolve in WFI or 10 mM NaCl, pH 6.8–7.2 to 0.2–1.0% w/v.
• Decolorize (optional): 0.1–0.5% activated carbon, 30 min, then depth filter.
• UF/DF: PES TFF cassettes 300–500 kDa MWCO; concentrate + 5–10 DV diafiltration with WFI (conductivity <100 µS/cm). Keep TMP ≤1.0 bar, low crossflow to protect MW.
• Endotoxin removal (Verify by LAL)
• MW banding (if required): Controlled ethanol titration or SEC-style fractionation.

Sterilization Strategy

• Preferred (solutions ≤0.5% w/v high-MW): 0.22 µm sterile filtration into sterilized vessel, then aseptic processing/fill.
• Powder API: Dry the precipitate (below) and gamma sterilize (validate MW impact), or compound aseptically later.
• Avoid moist heat on solution (depolymerizes HA).

Drying, Milling, Packaging (API option)

• Vacuum/lyophilization at ≤40 °C (powder moisture ≤10%).
• Low-energy, cooled milling → target mesh.
• Pack under nitrogen, double pharma PE liners + foil drum. Control RH, temp.

Quality Control (release)

• Identity(FT-IR, ¹H-NMR; enzymatic fingerprint)
• HA % Assay (CPC titration / HPAE-PAD, ≥98–99%)
• MW, Đ (GPC-MALS / intrinsic viscosity)
• Protein (BCA/Bradford ≤0.1%)
• Nucleic acids (A260/Fluorescent Dye ≤0.05%)
• Endotoxin (LAL ≤0.5 EU/mg, or tighter)
• Residual solvents: GC (ethanol/IPA) ≤0.5% total
• Metals: ICP-MS (USP/EP limits)
• Bioburden (USP <61>/<62>)
• pH (1% soln): 6.0–7.5
• Loss on drying (105 °C/KF) ≤10%
• Appearance: White–off-white