Hyaluronic Acid Purification Process
HA from Streptococcus fermentation — cell removal, protease treatment, activated charcoal, ethanol precipitation, and freeze-drying for cosmetic and pharmaceutical grades
Process Overview
Hyaluronic acid (HA) is a high-molecular-weight polysaccharide (500 kDa–3 MDa) secreted extracellularly by Streptococcus equi or Streptococcus zooepidemicus in fed-batch fermentation. The downstream challenge is removing bacterial cells, proteins, pigments, and endotoxins while preserving the native molecular weight and viscoelastic properties. The process combines physical clarification, enzymatic protein removal, adsorption, and polymer precipitation. Overall yield: 60–75% with >95% HA purity.
<0.5 EU/mg
Endotoxin (cosmetic)
Process Steps
1
Clarification
Cell Removal: Centrifugation + Microfiltration
After fermentation, cells are removed by disc-stack centrifugation (8,000–12,000 × g). The viscous HA broth requires dilution (1:2 with water) before centrifugation to reduce viscosity. The centrate is then polished by tangential-flow microfiltration (0.2 μm TFF) to achieve <100 CFU/mL bioburden. HA is fully retained in the permeate of MF; cells and cell debris are retained in the retentate.
Yield: >95%
Cell removal: >99.9%
2
Enzymatic Treatment
Protease Treatment (Pronase at 50°C)
Add Pronase (broad-spectrum protease, 0.1–0.5 mg/g HA) to the clarified broth and incubate at 50°C for 2 hours at pH 7.5. Pronase digests residual bacterial proteins and glycoproteins to small peptides and amino acids. Heat inactivate the protease at 80°C for 20 min. This step reduces total protein content from ~5% to <0.5% of HA mass, critical for endotoxin reduction and final purity.
Yield: >98%
Protein reduction: >90%
3
Adsorption
Activated Charcoal Treatment
Add activated charcoal (0.5% w/v, food-grade) to the protease-treated solution and stir at 25°C for 30 minutes. Charcoal adsorbs pigments, melanoidins, and hydrophobic contaminants that cause yellow discoloration. Filter charcoal through 0.45 μm membrane or filter press. The resulting HA solution should be colorless to faintly yellow (OD420 < 0.2). Charcoal dose optimization is critical: excess charcoal can also adsorb HA.
Yield: 90–95%
Color removal: >80%
4
Precipitation
Ethanol Precipitation
Add 3 volumes of cold 95% ethanol (final concentration ~75% v/v) to the HA solution while stirring. HA precipitates as white fibrous strands due to reduced water activity (polysaccharides are insoluble in high-ethanol mixtures). Collect precipitate by centrifugation (3,000 × g, 15 min) or sedimentation. Wash precipitate once with 70% ethanol to remove residual salts and small molecules. The precipitate contains >95% HA.
Yield: 85–92%
Purity: >95%
5
Polishing
Redissolution + Sterile Filtration
Redissolve HA precipitate in water or 0.9% NaCl (1–5 mg/mL). Filter through 0.22 μm PES or PVDF membrane for bioburden control. The high viscosity of concentrated HA solution requires pressure filtration (0.5–2 bar). Test endotoxin by LAL assay (<0.5 EU/mg for cosmetic, <0.1 EU/mg for ophthalmic), and measure molecular weight by SEC-MALS or viscometry.
Yield: >98%
Endotoxin: <0.5 EU/mg
6
Drying
Freeze-Drying
Freeze-dry (lyophilize) the sterile HA solution to produce a white fluffy powder. Lyophilization preserves the native molecular weight and avoids thermal degradation (HA degrades above 70°C). Typical cycle: freeze to −40°C, primary drying at −20°C / 0.1 mbar, secondary drying at 20°C. Final moisture content: <5% w/w. The powder is stable for 2–3 years at room temperature when sealed under nitrogen.
Yield: >99%
Form: Lyophilized powder
Target Molecule: Hyaluronic Acid
| Molecular Weight | 500 kDa – 3 MDa (fermentation-dependent) |
| Charge | Strongly anionic (one carboxylate per disaccharide repeat) |
| Solubility | Highly soluble in water; insoluble in >70% ethanol |
| Structure | Repeating disaccharide: N-acetylglucosamine – glucuronic acid |
| Viscosity | Extremely viscous at >1 mg/mL; pseudo-plastic behavior |
| Applications | Dermal fillers, ophthalmic viscoelastics, joint injection, cosmetics |
Cost Considerations
| Step | Key Cost Driver | Relative Cost |
|---|
| Centrifugation + MF | Centrifuge energy, MF membrane replacement | Medium |
| Protease Treatment | Pronase enzyme, incubation tanks | Medium |
| Activated Charcoal | Food-grade charcoal, filter press | Low |
| Ethanol Precipitation | Ethanol solvent recovery/recycling, centrifuge | High |
| Sterile Filtration | 0.22 μm capsules, high-pressure housing | Medium |
| Freeze-Drying | Lyophilizer capital cost, energy, cycle time | High |
Ethanol recovery and lyophilization dominate operating costs. Ethanol distillation/recovery systems reduce solvent costs by 80–90%. Spray-drying can replace lyophilization for lower-grade applications, significantly reducing cost. Use
untangle.bio to compare drying method economics.
Frequently Asked Questions
Why is Streptococcus used for HA fermentation instead of animal tissue extraction?
Streptococcus fermentation produces HA without the risk of animal-derived pathogens (viruses, prions) present in rooster combs or umbilical cords. The fermentation process also allows control of molecular weight through agitation, pH, and dissolved oxygen conditions. Modern fermentation achieves 6–8 g/L HA titers, making it economically competitive with extraction-based processes.
Why is molecular weight important for HA applications?
High molecular weight HA (>1 MDa) provides superior viscoelastic properties for dermal fillers and joint injections. Low molecular weight HA (10–100 kDa) is used for wound healing and penetrates skin more readily for topical applications. Ultra-low MW HA (<10 kDa, oligomers) has different biological activities and can be pro-inflammatory. Each application specifies a target MW range that the purification process must preserve.
How is endotoxin removed from fermentation-derived HA?
Endotoxin (lipopolysaccharide from Gram-negative bacteria) in HA is addressed by: (1) using endotoxin-free Streptococcus strains, (2) protease treatment (degrades lipid A protein associations), (3) activated charcoal (adsorbs some endotoxin), and (4) ultrafiltration (some endotoxin >100 kDa). For injectable-grade HA (<0.1 EU/mg), additional anion exchange chromatography (endotoxin binds to AEX resin) or Mustang Q filtration may be needed.
Can ethanol precipitation be replaced with membrane-based purification?
Yes. Ultrafiltration with a 100–300 kDa MWCO membrane concentrates HA while passing small molecules (salts, sugars, peptides). Diafiltration with 5 diavolumes of water removes >99% of small molecular impurities. This avoids ethanol handling and preserves molecular weight better. However, membrane fouling by HA is a challenge; cross-flow TFF systems are required. Membrane-based purification is increasingly preferred for injectable-grade HA.
Design Your Hyaluronic Acid Purification Process
Simulate the full HA downstream train from clarification through freeze-drying, model mass balance, and compare ethanol precipitation vs membrane filtration economics.
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