A Comprehensive Breakdown of the Full Production Process for Biologically Fermented Sodium Hyaluronate

Sodium hyaluronate (hyaluronic acid)—an essential raw material for skincare, dermal fillers, orthopedics, and ophthalmology—has long since moved beyond the era of scarcity when it was extracted from chicken combs and bovine eyes. Today, over 82% of the global supply is produced via microbial biosynthesis, underpinned by a comprehensive suite of cutting-edge biomanufacturing technologies, including Streptococcus fermentation, synthetic biology modifications, and precise molecular weight control. This article provides a comprehensive breakdown of the entire industrial-scale biosynthesis process, compares the gaps between the three generations of production technologies, and analyzes the core technical barriers in hyaluronic acid production.

I. Three Generations of Production Technology Iterations: Biosynthesis Replaces Traditional Methods Through Technical Superiority

Historically, there have been three routes for the preparation of sodium hyaluronate, and the differences in their merits and demerits directly determine the safety, cost, and maximum efficacy of the raw material.

First-Generation Animal Tissue Extraction Method: In the early days, this method relied on extraction from chicken combs and bovine vitreous humor. Raw material supply was limited, and the extraction yield was less than 0.5%. Its most critical flaw was the risk of residual heterologous proteins, viruses, and hyaluronidase. Injectable-grade products were highly prone to causing redness, swelling, and immune rejection. Currently, this method is used only in very small-scale laboratory settings and has been completely phased out of the industrial market.

Second-generation chemical total synthesis: This method relies on multi-step glycosylation protection and deprotection reactions to assemble disaccharide units. The entire process involves highly toxic organic solvents, with a total yield of less than 25%. It is suitable only for milligram-scale oligosaccharide laboratory research and development and cannot support mass production at the 10,000-metric-ton scale. The cost of high-purity medical-grade synthetic products is dozens of times that of the fermentation method, rendering it commercially unviable on a large scale.

Third-generation microbial biosynthesis (industry standard): Utilizing Streptococcus epizooticus and genetically engineered Bacillus subtilis as cell factories, and inexpensive carbon sources such as glucose and corn sugar as raw materials, the intracellular enzyme system of the microbial cells autonomously synthesizes the two major precursors—UDP-glucuronic acid and UDP-N-acetylglucosamine, two key precursors, which are then alternately polymerized into long-chain HA by hyaluronan synthase (HAS). The entire process involves mild, aqueous-phase reactions, is free of viral or heterologous impurities, and allows for precise control of molecular weight. Scaled-up fermentation yields can reach 10 g/L, with an overall yield exceeding 63%, making it the optimal solution for the cosmetics, pharmaceutical, and food industries.

The new generation of synthetic biology-engineered strains is at the forefront of the industry: by knocking out the genes encoding hyaluronidase and proteases in the bacterial cells and enhancing HAS expression, yield has increased by more than 40%, while endotoxin levels have been significantly reduced, making it suitable for the production of high-end, injectable-grade filler raw materials.

II. Complete Industrial Biosynthesis Process Flow: Six-Stage Process for Precise Control of Molecular Weight and Purity

1. Domestication and Preservation of Genetically Engineered Strains (Upper Limit of Production Capacity at the Source)

The first critical step in mass production is the construction of high-yield, safe strains. There are two mainstream industrial strains: natural Streptococcus equi and GRAS-certified engineered Bacillus subtilis. During R&D, gene editing is used to enhance the hasA, hasB, and hasC synthesis pathways and to knock out the HA hydrolase gene, thereby preventing the breakdown and degradation of long-chain hyaluronic acid during fermentation. Strains are preserved using a liquid nitrogen storage system with dual-person, dual-lock protocols. Prior to production, triple verification—including sequencing, toxin production, and yield—is completed to prevent strain degradation or mutation. During the activation phase, composite nutrient agar plates are used for cultivation, and single colonies are selected for stepwise expansion in shake flasks to ensure uniform cell viability in the seed culture, which directly determines the stability of subsequent fermentation.

2. Stepwise Feed Addition in High-Density Deep-Tank Fermentation (Core Process Control Point)

The entire fermentation process is divided into three expansion stages: 5L shake flask seed culture → 50L intermediate seed tank → 1,000L-class sealed main fermentation tank, with a cycle of 42–48 hours. Five key parameters are precisely and dynamically controlled throughout the entire process.

3. Fermentation Broth Pretreatment: Sterilization and Impurity Removal

At the end of fermentation, the broth contains a mixture of bacterial cells, polysaccharides, and impurity proteins. The first step involves high-temperature inactivation of the bacterial cells to prevent their enzymes from continuing to degrade HA; subsequently, ceramic membrane microfiltration retains cellular residues. The rough filtrate is treated with activated carbon to adsorb pigments, hydrophobic impurity proteins, and trace endotoxins, thereby preliminarily purifying the crude product and avoiding excessive chromatographic load that could result in product loss during later stages.

4. Multi-Stage Membrane Separation for Concentration and Purification (Determines Raw Material Grade)

Cosmetic-grade products require only two stages of ultrafiltration for desalination and concentration; medical-grade raw materials for injection must undergo dual purification via series-connected nanofiltration and dialysis filtration to retain long-chain HA while filtering out small-molecule amino acids, inorganic salts, and nucleic acid impurities. High-end industrial production lines incorporate cation-exchange chromatography to thoroughly remove endotoxins and protein allergens; following chromatography, HA purity directly exceeds 99.5%, meeting the national standard limits for Class III medical device fillers. This represents the core technological difference between affordable skincare ingredients and medical aesthetics injectable ingredients.

5. Low-Temperature Ethanol Precipitation for Solid-Liquid Separation

The purified HA aqueous solution is introduced into a low-temperature ethanol system, causing sodium hyaluronate to precipitate in a controlled manner. Operating at 4°C prevents molecular chain breakage; the flocculent precipitate is collected by centrifugation, and residual organic solvents are removed through multiple ethanol washes. The entire process is conducted in a sealed, explosion-proof cleanroom to eliminate secondary contamination from heavy metals and microorganisms.

6. Aseptic Freeze-Drying, Pulverization, Inspection, and Packaging

The precipitate undergoes vacuum low-temperature freeze-drying to remove all moisture while preserving the intact long-chain structure. The powder is aseptically pulverized and sieved to different mesh sizes. The entire batch is tested for molecular weight, purity, endotoxins, microbial limits, and light transmittance. After passing inspection, the product is packaged in vacuum-sealed aluminum foil and categorized into three finished product classes—medical injection grade, medical aesthetics and skincare grade, and oral food grade—before being stored in the warehouse.

III. Four Overwhelming Advantages of the Biosynthetic Method, Underpinning the Global Monopoly of Domestic Products

1. Safe with Zero Xeno-Risk: The microbial process is entirely free of animal-derived impurities and poses no risk of cross-species viruses or prions. It carries zero risk of immune stress for sensitive skin or in injection applications, and meets the stringent market access standards for clean beauty products and medical devices in Europe and the United States.

2. Precise Molecular Weight Customization: By freely adjusting fermentation temperature and feed strategies, the process enables integrated production of long-, medium-, and low-molecular-weight HA as well as oligomeric HA. A single production line can simultaneously supply raw materials for the full range of products—including dermal fillers, serums, eye drops, and joint injections—a level of graded purification that extraction methods cannot achieve.

3. Scalable Cost Reduction and Unlimited Supply: Leveraging renewable corn-derived glucose as a feedstock, 10,000-metric-ton-scale fermentation facilities are in continuous operation. The average raw material price has dropped by 30% over the past five years. Leading domestic companies have achieved annual production capacities exceeding 400 metric tons per line, accounting for 60% of global production capacity, and export to high-end markets in Europe, the U.S., Japan, and South Korea.

4. Green, Low-Carbon, and Environmentally Friendly: The entire process utilizes aqueous-phase bioreactions, generating no large volumes of highly toxic organic hazardous waste. Waste liquid can be treated through simple neutralization, aligning with global trends in green raw material certification. Domestically produced fermented hyaluronic acid is suitable for vegan products and carries a “zero animal testing” label, commanding a premium among overseas brands.

IV. Existing Process Challenges and Cutting-Edge Industry Technology Directions

The current core challenge in fermentation is the difficulty of oxygen transfer in high-viscosity fermentation broth; insufficient dissolved oxygen limits production capacity when achieving high yields of long-chain HA. Additionally, trace endotoxins from natural Streptococcus bacteria require complex chromatographic removal. Two major cutting-edge breakthroughs in the industry: First, the genetic engineering of Bacillus subtilis to produce a strain that is non-toxic and endotoxin-free, simplifying downstream purification processes; second, the cell-free enzymatic co-synthesis technology, which synthesizes HA in vitro using a “one-pot” method involving eight enzymes, completely eliminating bacterial metabolic byproducts. This ultra-high-purity oligomeric HA is exclusively supplied for high-end medical aesthetics and skin repair products and is currently transitioning from pilot-scale testing to pilot-scale production.

Conclusion

Contrary to the common perception that hyaluronic acid is merely a moisturizing ingredient, the underlying biomanufacturing technology directly determines the raw material’s purity, molecular weight, and safety. In high-end medical applications such as aesthetic fillers, ophthalmology, and orthopedics, only high-purity HA produced through rigorous biological fermentation can meet clinical standards. In the future, as synthetic biology continues to drive innovation, customized modified HA and oligomeric hyaluronic acid derivatives will continue to open up new market segments. It is now a foregone conclusion that domestically produced biosynthetic raw materials will dominate the global market landscape in the long term.

From the hyaluronic acid synthesis reaction to subsequent purification of the reaction mixture and concentration of the raw solution, comprehensive hardware infrastructure is key to ensuring stable product quality. We provide process equipment—including reactors suitable for hyaluronic acid synthesis and multi-stage membrane separation and concentration systems—and offer flexible solutions tailored to different molecular weights and production capacity requirements. We empower the R&D and mass production of hyaluronic acid raw materials and work together to advance the high-quality development of the biopolysaccharide industry.