Hyaluronic Acid (HA), also known as hyaluronic acid, is a glycosaminoglycan composed of disaccharide units of D-glucuronic acid and N-acetylglucosamine. It is a natural moisturizing factor in the human body that can absorb and retain large amounts of water, increasing tissue moisture content, thus improving skin hydration and moisture retention. Hyaluronic acid is widely used in various cosmetic products, as well as in medical aesthetics for wrinkle removal and facial filling.

Hyaluronidase (HAase) is a class of enzymes that degrade hyaluronic acid by acting on the β-1,4 glycosidic bonds in the hyaluronic acid sugar chain, thereby catalyzing the hydrolysis of hyaluronic acid, reducing its viscosity, and improving tissue permeability.

Types of Hyaluronidase

Hyaluronidase can be categorized based on its source into animal-derived hyaluronidase and recombinant human hyaluronidase:

– Animal-derived Hyaluronidase: Mainly extracted and purified from animal (cow or sheep) testicular tissue. Due to the complexity and diversity of its sources, it can easily cause allergies, immunogenicity, and even zoonotic infectious diseases.

– Recombinant Human Hyaluronidase: The human genome contains six types of hyaluronidase: HYAL-1, HYAL-2, HYAL-3, HYAL-4, HYAL-P1, and PH-20. Among them, HYAL-P1 is a pseudogene; HYAL-3 shows no enzymatic activity; HYAL-4 has almost no activity towards hyaluronic acid; HYAL-1, HYAL-2, and PH-20 can degrade hyaluronic acid. HYAL-1 is a prototype acidic active enzyme with an optimal pH of about 3.8; HYAL-2 is an acidic active enzyme with an optimal pH of about 5.6; PH-20 is a prototype neutral active enzyme with the strongest biological activity among hyaluronidases in the human body, being the primary enzyme responsible for degrading HA in the human body and the only hyaluronidase active under neutral conditions (optimal pH 5-7), making it most suitable for the physiological environment in the body.

Hzymes Biotech recombinant hyaluronidase is a human-derived hyaluronidase (rHuPH20) expressed using Chinese hamster ovary (CHO) cells through recombinant DNA technology. It undergoes a high-purity preparation process and strict quality control, resulting in higher purity, superior biological activity, lower endotoxin levels, stricter batch-to-batch control, and no animal-derived or exogenous viral contamination.

Applications of Hyaluronidase

– Subcutaneous Injection:

Traditional antibody drugs are usually administered via intravenous injection. However, subcutaneous injection, which shortens administration time, allows fixed doses regardless of patient weight, reduces dosage control costs, and enables at-home self-administration, has gradually become a new research direction for antibody drug formulations. Currently, among the FDA-approved antibody drug administration routes, subcutaneous injection is the second most common after intravenous infusion.

The key challenge of subcutaneous injection is the limitation of the injection volume, generally requiring a volume of less than 2 mL to avoid pain and hardening at the injection site. This necessitates the preparation of high-concentration (over 100 mg/mL or even higher) protein formulations. However, developing high-concentration protein solutions involves issues such as high protein viscosity, protein solubility, protein degradation and aggregation, and protein stability during long-term storage.

Recombinant human hyaluronidase (rHuPH20) can temporarily degrade hyaluronic acid locally to enhance the dispersion and permeability of drugs within tissues, thereby increasing drug bioavailability and enabling the conversion from intravenous to subcutaneous administration and dose optimization.

Currently, there are four approved antibody drug products and one approved immunoglobulin product that incorporate hyaluronidase.

Mechanism of Action:

The mechanism of action of recombinant hyaluronidase (rHuPH20) involves locally depolymerizing HA in the subcutaneous space to facilitate rapid drug flow, thereby temporarily removing barriers to larger volume subcutaneous injections. Specifically:

• Hyaluronic acid creates resistance to large fluid flow, limiting subcutaneous drug delivery, dispersion, and absorption;
• Hyaluronidase depolymerizes hyaluronic acid;
• This promotes large fluid flow and increases the dispersion and absorption of co-administered therapeutic agents.

Within 24 hours after injection, hyaluronidase restores normal interstitial viscoelasticity without inflammatory changes or histological alterations. The impact on the structure of the subcutaneous space is temporary and reversible. Hyaluronidase has good tolerability, with over 20 clinical studies examining its immunogenicity, none of which detected neutralizing antibody activity against hyaluronidase. Compared to intravenous administration, no clinically significant differences in immunogenicity were observed with subcutaneous administration of antibody drugs.

Immunotherapy:

 CAR-T immunotherapy has shown unique advantages in cancer treatment, particularly for hematological malignancies, but its efficacy against solid tumors remains limited. Solid tumors have a dense extracellular matrix (ECM) rich in collagen and hyaluronic acid, which hinders T cell infiltration into deep tumor regions. Additionally, the tumor immunosuppressive microenvironment plays a significant inhibitory role in CAR-T therapy. Therefore, coupling hyaluronidase (rHuPH20) and α-PDL1 (PD-L1 antibody) to CAR-T cells can enhance tumor penetration while also blocking immune checkpoints, showing enhanced solid tumor infiltration and growth inhibition in both in vitro and in vivo experiments.

When constructing CAR-T cells with lentivirus, a metabolic glycoprotein marker, N-azidoacetylmannosamine tetraacetate (Ac4ManNAz), is added to the culture medium, and the azide group is incorporated into the T cell membrane through intrinsic biosynthesis. Azide-modified cells are then conjugated with dibenzocyclooctyne (DBCO)-modified hyaluronidase (rHuPH20) to enhance tumor penetration, while DBCO-modified α-PDL1 is conjugated with a tumor cell-extracellular acid-responsive maleimide bond. α-PDL1 can be released from α-PDL1- and HAase-engineered cells under the acidic conditions of the tumor microenvironment, reversing the PD1-PDL1 pathway-mediated immunosuppression and thereby enhancing antitumor activity.

Medical Aesthetics:

In medical aesthetics, macromolecular hyaluronic acid (hyaluronic acid) is widely used for injection filling and sculpting due to its high hardness. However, improper facial hyaluronic acid injections can lead to various adverse reactions, the most severe of which are complications caused by vascular embolism after injection, potentially leading to skin tissue ulceration, ischemic necrosis, vision loss, or even blindness.

Hyaluronidase (rHuPH20) is often used to treat complications following hyaluronic acid injection procedures.

Procedure Reference:

The patient lies in a supine position, and under color Doppler ultrasound, the side artery is located approximately 1 cm anterior to the mandibular angle, marked as the puncture point. After facial disinfection, local anesthesia is administered at the puncture point. An arterial blood sampling needle or disposable intravenous infusion needle is used to puncture the artery through the skin, with the needle angled at 30°-45° relative to the skin. The needle is slowly advanced, and when blood is observed in the needle core, it is pushed forward another 2-3 mm. Once pulsatile blood flow is seen at the tail of the needle, it indicates successful arterial puncture. After confirming successful puncture, a hyaluronidase solution (1000 U/mL) is slowly injected into the facial artery, with an injection volume ranging from 0.5 to 1.5 mL.

Assisted Reproduction:

Common assisted reproductive technologies include in vitro fertilization (IVF)—where sperm are adjusted to an appropriate concentration and combined with an egg for fertilization—and intracytoplasmic sperm injection (ICSI)—where a single sperm is directly injected into the egg cytoplasm under a microscope to achieve fertilization.

Before intracytoplasmic sperm injection into oocytes, the cumulus and granulosa cells must be removed from the oocytes to expose the oocyte zona pellucida for injection. Brief exposure of the oocyte clusters to recombinant hyaluronidase solution can hydrolyze the hexosamine bonds in the hyaluronic acid that binds cumulus and granulosa cells together, allowing granulosa cells to be separated from the oocytes, making the oocytes suitable for intracytoplasmic sperm injection.

Product Overview and Performance

– In Vivo Biological Activity:

Subcutaneous injection of different doses of Hzymes BiotechNew Enzyme’s hyaluronidase shows high in vivo biological activity, with superior diffusion effects compared to foreign manufacturers’ products.

– Product Stability:

Hzymes Biotech hyaluronidase enzyme powder and solution exhibit high thermal stability and remain stable after multiple freeze-thaw cycles.

Product Ordering

CHO Recombinant Expression Human Series	HBP000121-01	1mg
	HBP000121-02	5mg
	HBP000121-03	10mg

Reference:

[1] Locke, Kenneth Wetal. “ENHANZE® drug delivery technology: a novel approach to subcutaneous administration using recombinant human hyaluronidase PH20.” Drug delivery vol. 26,1 (2019): 98-106.

[2] Zhao,Yangyangetal. “Bioorthogonal Equipping CAR-T Cells with Hyaluronidase and Checkpoint Blocking Antibody for Enhanced Solid Tumor Immunotherapy.” ACS central science(2022),8,603-614.

[3] 付强, 周桂文 ,等. 经皮面动脉透明质酸酶注射治疗面部注射美容致血管栓塞的效果观察[J].华整形外科杂志,2021,37 (04): 365-370.

In the future, Hzymes biotech will always remember its original intention and persist in meticulous cultivation in the IVD field. It will adhere to independent research and development, accelerate the construction of a world-class specialty enzyme production platform, and achieve import substitution of core enzyme raw materials in the field of biomedicine in China. It will collaborate with leading biopharmaceutical companies to expand their global presence and contribute to the advancement of the industry.