On November 2, commercial insurance catalog negotiations officially began, with five CAR-T therapies entering the negotiation list: Fosun Kite’s Axicabtagene Ciloleucel, JW Therapeutics’ Relmacabtagene Autoleucel, IASO Bio’s Equecabtagene Autoleucel, CARsgen Therapeutics’ Zevo-Cabtagene Autoleucel, and Abogen’s Nacacabtagene Autoleucel.

As a supplement to the basic medical insurance system, the innovative drug catalog under commercial insurance adopts a negotiated pricing mechanism instead of deep price cuts, reserving space for the development of innovative therapies.

Since the beginning of this year, the biopharmaceutical industry has witnessed an intense “arms race.” AstraZeneca, AbbVie, Gilead, and BMS have each spent hundreds of millions of dollars to acquire early-stage in vivo CAR-T companies. In sharp contrast, traditional autologous and allogeneic (off-the-shelf) CAR-T therapies are showing growth bottlenecks — complex manufacturing, high cost, and limited efficacy against solid tumors.

Schematic diagram of CAR lentiviral vector structure

Reconstructing the Treatment Logic: Precision Delivery and Cellular Engineering

Although traditional ex vivo CAR-T therapy shows great potential, it has always been limited by three major challenges:

Complex process: 

It requires blood collection, ex vivo T-cell modification and expansion, which are time-consuming and rely on specialized facilities.

Limited manufacturing capacity: 

The personalized nature of production makes large-scale manufacturing difficult, restricting patient accessibility.

Preconditioning dependency: 

Patients need to undergo chemotherapy to deplete immune cells before infusion, increasing infection risks.

In contrast, in vivo CAR-T delivers CAR genes directly into the body via specific carriers, precisely targeting T cells, NK cells, or even hematopoietic stem cells for in situ genetic modification. This strategy retains the precision killing power of CAR molecules while achieving scalability — breaking through the long-standing limitations of traditional CAR-T therapies.

Technical Routes:

“Long-acting” Lentiviral Vector VS “Controllable” LNP Vector

Lentiviral vectors – One-time treatment for long-term protection:

Lentiviral vectors integrate the CAR gene stably into the T-cell genome, enabling durable CAR expression and prolonged tumor-killing capability. The core technologies lie in T-cell–specific targeting, transduction efficiency, and safety regulation.

01. T-cell targeting transformation: from broad tropism to T-cell specificity

Traditional lentiviruses use broadly tropic envelope proteins (such as VSV-G), which can infect hepatocytes and endothelial cells. Dual engineering — detargeting + retargeting — achieves T-cell–specific delivery:

Envelope protein engineering: 

Replace VSV-G with a CD28L chimeric envelope, fusing the T-cell co-stimulatory ligand CD28L. This specifically binds to the CD28 receptor on T cells, increasing transduction efficiency 30-fold over wild-type lentivirus while reducing off-target hepatocyte infection to <0.1%.

Genomic element optimization: 

Introduce an RACR/CAR system where the lentiviral vector carries both a “reverse responsive element” and the CAR gene. Without drug administration, CAR expression remains silent and T cells are quiescent. After oral administration of the small molecule AP21967, the responsive element changes conformation to activate CAR expression, peaking in 48 hours and silencing again within 7 days after drug withdrawal — reducing CRS incidence by 60%.

02. Transduction efficiency optimization: high-efficiency T-cell modification at low doses

Lentiviral transduction efficiency depends on viral titer, T-cell activation, and co-stimulatory signals. The following strategies enable breakthroughs:

High-titer production: 

By adopting a “suspension + serum-free culture” system and optimizing HEK293T transient transfection, viral titers increased from 1×10⁸ TU/mL to 5×10⁹ TU/mL. This supports effective transduction at one-tenth the standard dose, minimizing off-target risks.

T-cell activation synergy: 

Embedding an IL-2 mimetic peptide within the vector activates T-cell proliferation signals, increasing the proportion of G1-phase T cells from 35% to 62%, boosting transduction efficiency 1.8×. Meanwhile, integrating 4-1BBL into the envelope provides a second co-stimulatory signal by binding 4-1BB on T cells, enhancing survival and persistence — with CAR-T cells persisting up to 12 months in cynomolgus monkey models.

03. Clinical safety regulation: enabling long-lasting T-cell killing

Hematologic malignancies — Sustained response at low dose: 

With CD45-ligand lentiviral delivery of CD19 CAR at 1×10⁸ TU/kg, peripheral T-cell transduction reached 63%, tumor burden dropped by 92% in lymphoma mouse models, and IL-6 levels were 50% lower than ex vivo CAR-T.

Solid tumors — Enhanced tumor penetration: 

Lentiviral vectors co-delivering “BCMA-CAR + TGFβ-neutralizing peptide” in multiple myeloma models neutralized TGFβ-mediated immunosuppression, enabling CAR-T infiltration deep into tumor cores. The Treg proportion in tumor microenvironment decreased from 32% to 11%, improving killing efficiency by 2.5×.

LNP Vector – “Safe, Controllable, and Flexible”

CAR mRNA does not integrate into the genome, naturally avoiding genotoxic risks. mRNA-LNP uses “antibody-targeted LNPs” to deliver CAR mRNA directly to T cells in vivo, achieving in situ T-cell reprogramming. The core technologies involve Ab-LNP targeting, formulation, and clinical validation.

01. Ab-LNP specific targeting: precise recognition of T cells

The key to Ab-LNP lies in antibody modification that forms a specific bridge between LNPs and T-cell receptors. The process includes: antibody conjugation of CD3/CD8-specific antibodies to LNP surfaces → specific binding to T-cell receptors → endocytosis and mRNA release → high-efficiency CAR expression within cytoplasm.

02. Ab-LNP formulation: enhancing T-cell transduction efficiency

Ab-LNP preparation optimizes antibody density, ionizable lipid pKa, particle size, and mRNA loading to maximize transduction efficiency.

03. Ab-LNP clinical validation: safety and efficacy

mRNA-LNP–based in vivo CAR-T technology has achieved multiple breakthroughs:

• Over 90% clearance of hematologic malignancies without hepatic toxicity;
• Significant symptom relief in autoimmune diseases (e.g., lupus) with no neurotoxicity;
• Effective solid tumor control by incorporating chemokine mRNA to improve infiltration — combined with PD-1 inhibitors, tumor suppression reached 89%.

Risks and Outlook: The “Last Mile” from Lab to Bedside

Although in vivo CAR-T holds great promise, several barriers remain before clinical translation:

Lentiviral long-term risks: 

Persistent CAR expression may cause chronic B-cell depletion; genomic integration risks require long-term monitoring.

mRNA-LNP short-lived expression: 

Transient CAR expression limits long-term tumor control, requiring repeat dosing.

Cell selectivity: 

Off-target transduction of non-intended cells can reduce efficacy or cause organ toxicity.

The industry is working to overcome these issues: lentiviral vectors are being optimized with improved envelope design to reduce immunogenicity and integration risks, while mRNA-LNP systems are leveraging circular mRNA to extend expression duration. Both are evolving toward more precise targeting and longer-lasting therapeutic effects.

In vivo CAR-T therapy marks a key evolutionary step in cell therapy — shifting from personalized, case-by-case treatment to standardized and scalable therapeutic models.
This technological revolution not only addresses the cost and accessibility challenges of traditional CAR-T therapy but also heralds a new era in cell-based immunotherapy.

Related Products

Bibliography

1.Du L, Nai Y, Shen M, Li T, Huang J, Han X, Wang W, Pang D, Jin A. IL-21 Optimizes the CAR-T Cell Preparation Through Improving Lentivirus Mediated Transfection Efficiency of T Cells and Enhancing CAR-T Cell Cytotoxic Activities. Front Mol Biosci. 2021 Jun 4;8:675179.

2.Dai Q, Han P, Qi X, Li F, Li M, Fan L, Zhang H, Zhang X, Yang X. 4-1BB Signaling Boosts the Anti-Tumor Activity of CD28-Incorporated 2nd Generation Chimeric Antigen Receptor-Modified T Cells. Front Immunol. 2020 Nov 13; 11: 539654. 

3.Xu J, Liu L, Parone P, Xie W, Sun C, Chen Z, Zhang J, Li C, Hu Y, Mei H. In-vivo B-cell maturation antigen CAR T-cell therapy for relapsed or refractory multiple myeloma. Lancet. 2025 Jul 19;406(10500):228-231. 

4.Andorko JI, Russell RM, Schnepp BC, Grubaugh D, Mullen KF, Wakabayashi A, Carrington LJ, O'Malley T, Kuri-Cervantes L, Culp TD, Johnson PR. Targeted in vivo delivery of genetic medicines utilizing an engineered lentiviral vector platform result in CAR T and NK cell generation. Mol Ther. 2025 Oct 1;33(10):4937-4952. 

5.Jacob Garcia, Christine Dehner, Jeffrey Teoh, Wayne Wallis, A Phase 1, Multicenter, Open-Label Study of UB-VV111 in Combination with Rapamycin in Relapsed/Refractory CD19+ B-Cell Malignancies, Blood, Volume 144, Supplement 1, 2024, Page 1750. 1, ISSN 0006-4971.

Among various preservation techniques, enzyme lyophilization (freeze-drying) stands out as a highly effective solution, offering enhanced stability and ease of storage. This article explores the science behind enzyme preservation and the key role that lyophilization technology plays in ensuring enzyme stability for industrial applications.

II. Enzyme Preservation: Why It Matters

Enzyme stability is critical for industries that rely on their catalytic properties, such as pharmaceuticals, food production, and bioenergy. If enzymes lose their activity during storage or transportation, the entire production process can be affected, leading to operational delays and financial losses. Additionally, the cost of replacing enzymes or maintaining a cold supply chain for liquid enzymes can be prohibitive.

The need for efficient enzyme preservation methods is therefore clear. Traditional methods, such as storing enzymes in liquid form under refrigeration, have limitations in terms of shelf life, ease of transportation, and cost. Lyophilization, which involves removing water from the enzyme solution to form a stable dry product, addresses these issues while preserving enzyme activity for long periods.

III. What is Lyophilization (Freeze-Drying)?

Lyophilization is a specialized process used to preserve sensitive biological materials, such as enzymes, by removing water through sublimation. In this process, an enzyme solution is first frozen at a low temperature, and then the ice is removed via a vacuum in a primary drying phase (sublimation), followed by secondary drying to remove any residual moisture.

The main advantage of lyophilization is that it allows enzymes to be stored in a dry, stable form that is easy to handle and transport. By preserving the enzyme in a solid form, lyophilization significantly extends its shelf life while maintaining its biological activity. This makes lyophilized enzymes ideal for long-term storage and global distribution, especially in industries where cold storage is not feasible.

IV. The Advantages of Enzyme Lyophilization

The lyophilization of enzymes offers a variety of advantages:

• Enhanced Stability: 

Lyophilized enzymes are far more stable than their liquid counterparts. By removing water, the enzyme’s molecular structure is protected, reducing the risk of denaturation or degradation that occurs in the presence of moisture or fluctuating temperatures.

• Longer Shelf Life: 

Lyophilized enzymes can be stored for extended periods (often years) without significant loss of activity, making them ideal for industries that require long-term enzyme storage or that need to keep inventory on hand for emergencies.

• Ease of Transport: 

Lyophilized enzymes do not require refrigeration, making them easier and less costly to transport. This is particularly important for global supply chains, where temperature-controlled logistics can be both expensive and complicated.

• Cost-Effectiveness: 

While the initial cost of lyophilization equipment may be high, the long-term savings in storage and transportation costs make lyophilized enzymes an economically viable solution for many industries.

V. Key Factors Affecting Enzyme Lyophilization

The lyophilization process must be carefully controlled to ensure enzyme stability. Several factors affect the success of enzyme freeze-drying:
 

• Enzyme Formulation: 

The addition of stabilizing agents, such as cryoprotectants or buffering agents, is critical in preserving enzyme activity during the freeze-drying process. These substances help to protect the enzyme’s structure from damage caused by freezing or dehydration.

• Lyophilization Conditions: 

The temperature, vacuum pressure, and drying time must be optimized to prevent denaturation. The freezing phase must ensure that the enzyme’s structure is preserved, while the drying phases need to be gentle to avoid destabilizing the enzyme’s active sites.

• Enzyme Structure: 

Enzymes with more complex structures or those requiring specific conditions for activity may be more challenging to lyophilize successfully. The choice of enzyme formulation and stabilizers plays a crucial role in overcoming these challenges.

VI. Lyophilization in Different Enzyme Applications

Enzyme lyophilization has proven to be beneficial in numerous industrial sectors:

• Pharmaceutical Industry: 

In pharmaceutical applications, lyophilized enzymes are used in drug formulations, diagnostics, and enzyme replacement therapies. The stability and portability of lyophilized enzymes ensure that they maintain their efficacy throughout the supply chain.

• Food Industry: 

Enzyme preparations for food processing, such as those used in brewing, dairy, or baking, are often lyophilized to maintain high activity levels without refrigeration. Lyophilization ensures that enzymes can be stored for extended periods, allowing for more efficient production processes.

• Bioenergy: 

Enzyme lyophilization also plays a role in biofuels, where enzymes are required for biomass breakdown and fermentation. The ability to store and transport these enzymes in a stable, dried form ensures a continuous and efficient biofuel production process.

• Health and Beauty: 

Lyophilized enzymes are used in the health and beauty industry for various purposes, including skincare formulations. The ability to preserve enzymes in a potent form makes them valuable ingredients in a variety of cosmetic products.

VII. Advancements in Lyophilization Technology

The technology behind enzyme lyophilization has advanced significantly over the years. Some of the key innovations include:

• Advanced Freeze-Drying Equipment: 

Modern lyophilization equipment offers precise control over temperature, pressure, and drying times, resulting in better-preserved enzymes with higher activity retention.

• Enhanced Stabilizers: 

New stabilizing agents, including synthetic and natural protectants, have been developed to improve the success of lyophilization, especially for more fragile enzymes.

• Nanotechnology: 

Research into nanoencapsulation and other advanced materials has opened the door to even more efficient enzyme preservation, allowing for the encapsulation of enzymes in nanoparticles that protect them during lyophilization and enhance their stability after rehydration.

VIII. Challenges and Limitations of Enzyme Lyophilization

Despite its many advantages, enzyme lyophilization does have some challenges:

• Enzyme Denaturation: 

Even with optimal conditions, some enzymes may lose activity during the freeze-drying process. Identifying the best formulation and stabilizers is crucial to minimizing this risk.

• Rehydration Time: 

Some lyophilized enzymes may take longer to rehydrate than others, which can be a limitation for certain high-volume industrial processes.

• Cost of Technology: 

The initial cost of lyophilization technology and the need for ongoing maintenance can be prohibitive for smaller businesses, although the long-term benefits often outweigh the initial investment.

IX. Case Study: Successful Lyophilization of Enzymes

One real-world example of successful enzyme lyophilization can be seen in the pharmaceutical industry. HZYMES, a leading biotech company, developed a lyophilized enzyme solution for an enzyme replacement therapy used in the treatment of a rare metabolic disorder. By employing advanced lyophilization techniques, HZYMES was able to extend the shelf life of the enzyme by over 50% compared to previous methods. This not only reduced transportation costs but also improved patient access to the treatment across global markets.

Conclusion

Enzyme preservation and lyophilization technology are critical components of the biotechnology and industrial sectors. Lyophilization offers significant advantages in terms of enzyme stability, ease of transport, and cost-effectiveness. As the demand for high-quality enzymes continues to grow across diverse industries, advancements in freeze-drying technology will further enhance enzyme preservation, allowing for more efficient and sustainable production processes.

For companies like HZYMES, embracing and advancing enzyme lyophilization technology ensures that enzymes remain potent, accessible, and ready for use, driving innovation and success in a variety of industries.

No complex instruments. No repetitive manipulation. Just one plasmid—and your target gene can undergo continuous hypermutation inside living cells, enhancing protein activity by thousands of times within a week!

It may sound like a synthetic biologist’s dream, but it has now become reality. On August 7, 2025, Peter Schultz’s team at the Scripps Research Institute published a groundbreaking study in Science, unveiling a system called T7-ORACLE, an orthogonal replication system that enables continuous, rapid, and targeted gene hypermutation in E. coli. This represents a revolutionary breakthrough in protein-directed evolution.

Bottlenecks in Traditional Evolution Technologies

Directed evolution is a key strategy for optimizing protein function, yet traditional approaches such as error-prone PCR or DNA shuffling have significant limitations: they rely on multiple rounds of in vitro mutation–transformation–screening cycles, which are labor-intensive and time-consuming, and they often fail to achieve deep evolutionary changes.

Although recent in vivo evolution technologies (like PACE) have improved efficiency, they still face issues such as non-targeted mutations, host genome instability, and the need for specialized equipment.

Inspired by bacteriophage T7, the Schultz team developed a fully orthogonal replication system composed of multiple components: T7 replicative proteins, including RNA polymerase, DNA polymerase, helicase–primase fusion protein, and single-stranded DNA-binding protein.

Mechanism:

The system works by expressing T7 RNA polymerase, DNA polymerase, helicase–primase, and single-stranded binding protein, which specifically recognize and replicate circular target plasmids carrying the T7 replication origin.

To promote replication, a catalytically defective T7 lysozyme is fused to the T7 RNA polymerase. The inhibitory effect of T7 lysozyme prevents full transcription, generating short RNA primers that initiate replication by T7 DNA polymerase.

By introducing engineered T7 DNA polymerase variants (Δ28 deletion and key mutations N520M, P560V, and V443K), the fidelity of replication is drastically reduced.

Figure 1. Schematic diagram of the directed evolution workflow

T7-ORACLE: A Precise “Genetic Editing Engine”

The Schultz team designed the T7-ORACLE system as a fully orthogonal replication mechanism inspired by bacteriophage T7.

It consists of several key components: T7 RNA polymerase, DNA polymerase, helicase–primase fusion protein, and single-stranded binding protein.

The system specifically targets plasmids carrying the T7 replication origin (φOR) for replication.

Figure 2. Life cycle of T7 bacteriophage

Figure 3. Schematic diagram of system design principle

To maximize mutational efficiency, the engineered DNA polymerase exhibits reduced proofreading activity and base selectivity, resulting in controlled hypermutation.

Most importantly, this system targets only the plasmid-borne genes without disturbing the host genome, allowing E. coli cells to maintain normal growth while achieving extremely high mutation rates in the target sequence.

How Is a Million-Fold Increase in Mutation Rate Achieved?

The researchers used rational design and directed evolution to iteratively optimize T7 DNA polymerase:

Removing proofreading activity: 

An exonuclease-deficient mutant (Δ28) raised the mutation rate 25-fold.

Reducing base-pairing fidelity: 

Key mutations N520M, P560V, and V443K further lowered replication accuracy.

Figure 4. Engineered T7 DNA polymerases involved in this study

The resulting five-mutation polymerase achieved a mutation rate of 1.7×10⁻⁵ per base per generation, nearly 100,000 times higher than the host genome mutation rate, while maintaining high transformation efficiency (2.4×10¹⁰ cfu/μg).

Stability tests of the optimized system included:

Fluctuation analysis, 

 confirming excellent orthogonality (genomic mutation rate 4.4×10⁻¹⁰ spb);

Sequencing of mutation spectra, 

showing unbiased substitution patterns (balanced transition/transversion rates);

Serial passage experiments, 

confirming plasmid stability and negligible metabolic burden for plasmids up to 13 kb

Figure 5. Stability testing of the optimized system

Application: Hyperactive β-Lactamase Evolved in Just One Week

To validate the system’s practicality, the team applied it to evolve TEM-1 β-lactamase.

In less than one week, mutants exhibiting 5,000-fold higher resistance to several clinically important antibiotics (aztreonam, cefotaxime, ceftazidime, and cefepime) were obtained.

Remarkably, the mutations that emerged (such as G238S, R164H, and E104K) were identical to those observed in clinical antibiotic-resistant strains, proving that the system accurately mimics natural evolutionary processes.

Figure 6. Continuous evolution of β-lactamase

Breaking Local Optima: From a Single Starting Point to Global Library Evolution

One of T7-ORACLE’s major advantages is its compatibility with pre-constructed mutation libraries.

The researchers compared different evolutionary starting points:

From the wild-type sequence 

– the evolution pathway was highly convergent (single-directional evolution).

From single-point saturation mutants 

– multiple unidirectional trajectories emerged.

From a full saturation library 

– multiple multidirectional trajectories were observed, enabling exploration of the global fitness landscape and identification of global optima.

Figure 7. Exploring broader fitness landscapes from multiple starting points toward whole-library evolution

Technological Prospects and Significance

Key advantages of this work include:

Development of a hypermutation system combining strong target specificity, safety, and long mutational reach 

— maintaining host genome stability while enabling evolution of any target protein length.

Accelerating protein functional evolution and overcoming local fitness peaks 

— by integrating pre-diversified mutation libraries to shift from directed evolution to global adaptive landscape exploration.

However, questions remain regarding whether the mutation distribution along target genes is fully uniform, and whether bias toward promoter-proximal regions occurs.

Note:

This study was published in Science on August 7, 2025, under the title:
“An orthogonal T7 replisome for continuous hypermutation and accelerated evolution in E. coli.”

Original article link:

https://www.science.org/doi/10.1126/science.adp9583

Product Recommendation

GMP-Grade T7 RNA Polymerase

During in vitro transcription, T7 RNA polymerase binds to the T7 promoter sequence in the DNA template to amplify a large amount of mRNA. Hzymes Biotech provides wild-type and more than 30 engineered T7 RNA polymerase mutants to meet the needs of different application scenarios.
 

Features

• High Fidelity: 

no significant difference in fidelity compared to wild-type T7 RNA by Sanger sequencing

• Strict QC panel: 

multi-dimensional quality control of enzymes; CV≤15%

• 30+ standard mutants available: 

High mRNA Integrity Mutant: Cat No.HBP000330 /Low dsRNA Mutant13: Cat No.HBP000340/Thermostable Mutant: Cat No.HBP000350

• GMP-Grade, Non-Animal Origin (AOF), DMF Record: MF036856

Orderings

Cell culture is at the heart of modern life science, from drug discovery to vaccine production and regenerative medicine. To achieve consistent, reliable results, researchers need reagents that are not only effective but also safe and reproducible. Traditional porcine-derived trypsin has long been used for cell dissociation, but it presents challenges such as variability, contamination risks, and regulatory concerns.

Hzymes’ Trypsin, Recombinant Digestion (TrypLE-like, GMP Grade) is an animal-origin-free recombinant enzyme for dissociating a wide range of adherent mammalian cells, including CHO, HEK 293, A529, primary human keratinocytes, and embryonic stem cells.

What is Recombinant Trypsin Digestion?

Recombinant trypsin digestion is a genetically engineered enzyme expressed in E. coli. Unlike traditional trypsin extracted from porcine pancreas, recombinant trypsin offers exceptional purity, consistency, and freedom from animal-derived components.

Hzymes developed its GMP-grade recombinant trypsin digestion reagent to meet the needs of biopharmaceutical manufacturing and advanced cell research. As a TrypLE-like alternative, it is optimized for both laboratory-scale and industrial applications, ensuring seamless integration into existing protocols.

Product Feature

1.     Gentle on cells—maintain cell health for reproducible results

Trypsin, Recombinant Digestion, like trypsin, cleaves peptide bonds on the C-terminal sides of lysine and arginine. However, Trypsin, Recombinant Digestion's exceptional purity increases specificity due to the action of a single enzyme. This reduces damage caused by cleavage from multiple enzymes in trypsin and other extracts.

2.     Easy to use—substitutes directly into existing protocols, with no inactivation required

Dilution alone inactivates trypsin and recombinant digestion, avoiding the need for trypsin inhibitors, such as FBS.

3.     Animal origin free (AOF)—pure, consistent, and effective

Activity unit: 25℃, pH 7.6, reaction system 3.2ml (1cm optical path). One trypsin unit (USP) is defined as the amount of trypsin that increases absorption value by 0.003 under 253nm through enzymolysis of BAEE per minute.

Applications in Biopharmaceuticals and Cell Research

Hzymes’ recombinant trypsin digestion (TrypLE-like) is versatile across cell culture and bioprocessing workflows:

CHO cell dissociation: 

Ideal for stable cell lines in protein expression and monoclonal antibody production

HEK293 cells: 

Reliable for viral vector production, vaccine development, and recombinant protein research

Stem cell dissociation: 

Gentle and reproducible, supporting embryonic stem cells and iPSC culture

Primary keratinocytes and A549 cells: 

Preserves morphology and viability for downstream assays

Drug discovery and diagnostics: 

Facilitates high-quality cell preparation for screening and research

Why Choose Hzymes?

Hzymes is a trusted provider of enzyme solutions for molecular biology, diagnostics, and biopharmaceuticals. With deep expertise in enzyme engineering, GMP manufacturing, and AOF-certified reagents, Hzymes delivers products that enable:

Consistency 

across research and industrial-scale production

Regulatory compliance 

for clinical and manufacturing use

Sustainability 

through elimination of animal-derived components

By choosing Hzymes’ Recombinant Trypsin Digestion (TrypLE-like alternative), researchers and manufacturers gain access to a gentle, easy-to-use, and GMP-grade solution tailored for the future of cell culture.

Orderings

Conclusion

As cell-based research and biomanufacturing continue to expand, the need for safe, reproducible, and regulatory-compliant reagents grows stronger. Hzymes’ Recombinant Trypsin Digestion (TrypLE-like, GMP Grade) is the ideal choice for scientists and biopharma companies seeking a gentle, AOF, GMP-certified alternative to traditional trypsin.

With proven performance in CHO, HEK293, stem cells, and beyond, it empowers researchers to achieve high-quality results with confidence.

Study on Bacterial Endotoxin Detection Methods for mRNA Vaccines Based on Lipid Nanoparticle Delivery Systems

Published in:

Chinese Journal of Microbiology and Immunology, November 2025

Author Affiliations:

Respiratory Virus Vaccine Division, National Institutes for Food and Drug Control (NIFDC)
Zhuhai Lifanda Biotechnology Co., Ltd.

Published Article

Bacterial Endotoxin Detection Methods Using Recombinant Factor C (rFC) for LNP-mRNA Vaccines

Using the gel-clot limit test described in Part III of the 2020 Chinese Pharmacopoeia, the authors evaluated HSV-2, RSV, and VZV mRNA vaccines. Through optimization of sample dilution factors, diluents, and interference testing, suitable conditions for bacterial endotoxin detection were established.

In this study, Hzymes’ Recombinant Factor C (rFC) Endotoxin Assay Kit was used to validate the detection results.

Lipid Nanoparticle (LNP) Structure and Its Impact on mRNA Vaccine Endotoxin Testing

mRNA technology, known for rapid design, high production efficiency, and strong immunogenicity, has opened new avenues in vaccines, oncology, infectious diseases, and autoimmune disorders. The emergency use of COVID-19 mRNA vaccines accelerated its clinical validation and industrialization.

mRNA is delivered into the body via lipid nanoparticles (LNPs), which typically contain cationic lipids, cholesterol, phospholipids, and PEGylated lipids. These components encapsulate nucleic acids through electrostatic interactions.

LNP-mRNA vaccines are primarily formulated as injectables, and bacterial endotoxin testing is a critical quality-control requirement. Endotoxins, structural components of Gram-negative bacterial cell walls, consist of O-antigen, core polysaccharide, and lipid A. Even trace amounts (1–5 ng/kg body weight) can trigger severe adverse reactions such as fever, sepsis, DIC, or shock.

Since LNPs are formed by hydrophobic lipid self-assembly, endotoxins may bind to lipid components during detection via electrostatic or hydrophobic interactions. Studies have shown that endotoxins can be adsorbed by liposomes when tested with LAL reagents, causing interference. Diluting lipid concentrations can reduce endotoxin–lipid interactions, and adding divalent cations like magnesium may also suppress interference.

Structural Diagram of Endotoxin

Materials and rFC Testing Methods for Accurate Endotoxin Analysis in LNP-mRNA Formulations

A portion of the study utilized different batches of Hzymes’ rFC assay kits along with unmasking buffer and optimized dilution factors to eliminate LNP-related interference, establish an endotoxin detection method suitable for LNP-mRNA vaccines, and validate the results.

Test Samples

HSV-2, RSV, and VZV mRNA vaccines provided by Zhuhai Lifanda Biotechnology Co., Ltd.

Reagents

Recombinant Factor C assay kits (range: 0.005–5 EU/mL; batch numbers HH20250401R, HH20250101R, HH20240601R) provided by Hzymes Biotech Co., Ltd.

Instrument

SpectraMax i3x fluorescence microplate reader.

Detection Procedures

1.      Using the formula MVD = cL/λ (c = 1.0 mL/mL; L = 30 EU/mL; λ = 0.005 EU/mL), the maximum valid dilution (MVD) was calculated as 6000×. Based on preliminary data, samples were diluted 100× using a Ca/Mg unmasking buffer.

2.      Following the kit instructions, endotoxin standards were prepared at 5, 0.5, 0.05, and 0.005 EU/mL, with endotoxin-free water as the negative control.
Each standard, sample, and control (100 μL) was added to 96-well plates in duplicate, followed by 100 μL of substrate. The instrument was set to Ex 380 nm / Em 440 nm. Fluorescence was recorded at 0 h and after a 1 h incubation at 37°C. ΔRFU was calculated by subtracting initial fluorescence, and net ΔRFU was obtained by subtracting the negative control.

3.      Data Analysis: A linear fit was generated using log concentration (x-axis) and log net ΔRFU (y-axis). Spike recoveries were calculated as:
Recovery (%) = (C_spiked – C_sample) / 0.5 EU/mL × 100%
A recovery of 50–200% indicated no interference.

Validation

1.     Standard Curve Validation:

Three batches of rFC kits produced standard curves with R² = 0.999, 0.990, and 0.999, all ≥0.980, meeting Chinese Pharmacopoeia 2020 requirements. Negative controls were below the lowest standard point (0.005 EU/mL). These curves were therefore valid for endotoxin quantification.

2.     Spike Recovery and Sample Testing:

Three batches each of HSV-2, RSV, and VZV vaccines were diluted 100×. One set served as the sample group; the other was spiked to 0.5 EU/mL. Endotoxin concentrations and spike recoveries were calculated using the standard curve equations. Recovery within 50–200% indicated no interference. Qualified samples were then used to calculate final endotoxin content based on the dilution factor.

Endotoxin Testing Results: rFC Assay Performance, Recovery Rates, and LNP Interference Removal

Standard Curve Reliability:

The three kit batches yielded R² values of 0.999, 0.990, and 0.999, all exceeding 0.980. Negative controls remained below the lowest standard point, meeting the validity requirements of General Chapter 1143 (photometric method) in the 2020 Chinese Pharmacopoeia.

Spike Recovery and Detection Results:

Across HSV-2, RSV, and VZV vaccine samples, rFC-based endotoxin measurements showed spike recoveries between 79.5% and 102.4%, within the acceptable 50–200% range. Endotoxin levels were <30 EU/mL and consistent with results obtained by the gel-clot method.

Conclusion and Future Outlook for rFC Endotoxin Assays in LNP-mRNA Vaccine Quality Control

LNP samples may interfere with LAL-based endotoxin assays. Using the unmasking buffer in the rFC kit effectively eliminated this interference. The rFC method produced results consistent with the gel-clot method, supporting its reliability.

This study provides an effective strategy for establishing endotoxin testing methods for LNP-mRNA vaccines, ensuring accuracy by minimizing interference. With the advancement of endotoxin detection technologies, methods such as rFC offer strong complementary value to traditional LAL assays, providing more comprehensive quality-control capabilities for LNP-based mRNA vaccines.

Recombinant Factor C (rFC) Endotoxin Detection Kit – Ordering Information

To obtain the original PDF, please contact: https://www.hzymesbiotech.com/index/user/register?url=/
Please verify: Recombinant Factor C

As global health management becomes a national priority, obesity and diabetes have emerged as rapidly growing public health concerns. According to the World Health Organization, more than 2 billion people worldwide are overweight or obese, while China alone has over 140 million diabetes patients. Metabolic disorders caused by obesity have intensified the demand for solutions that combine healthy weight loss and blood-glucose management.

Against this backdrop, Semaglutide—a long-acting GLP-1 receptor agonist—has become the world’s most prominent metabolic-disease therapeutic. In 2024–2025, Semaglutide demand surged globally, and by Q1 2025, it even surpassed Keytruda to become the world’s best-selling drug. This explosive market demand has accelerated the transformation of manufacturing technologies, shifting from traditional chemical synthesis toward biosynthetic, fermentation-based production.

What Is Semaglutide?

Semaglutide is a GLP-1 analog widely used for weight management and type 2 diabetes treatment. By activating the GLP-1 receptor, it enhances insulin secretion, reduces appetite, slows gastric emptying, and helps patients effectively manage both body weight and blood glucose.

Its efficacy, safety profile, and long half-life have made it a leading molecule in the global metabolic-disease drug market.

Why Traditional Chemical Synthesis Is Limiting

Conventional chemical peptide synthesis requires multiple protection, coupling, and deprotection steps. Challenges include:

Low recovery rate

High production cost

Long manufacturing cycle

Complex impurity control

As a result, the industry is increasingly adopting biosynthetic methods, which offer higher yields, better scalability, and improved cost-effectiveness for long peptides like Semaglutide.

Biosynthesis: A Breakthrough in Semaglutide Manufacturing

Modern biosynthetic Semaglutide uses prokaryotic or eukaryotic expression systems to generate the core peptide chain via fermentation. The process generally includes:

Expression of a precursor peptide fused with a solubility-enhancing tag

Extraction and purification from fermentation broth

Enzymatic removal of the tag

Chemical conjugation of the N-terminal non-proteinogenic amino acid

Final purification to obtain high-purity Semaglutide

This method significantly shortens production timelines and reduces cost while maintaining high quality and consistency.

HCD/HCP Risk Control: A Critical Quality Requirement

Target peptides in fermentation lysates coexist with impurities such as host proteins, host nucleic acids, and endotoxins. The mainstream purification strategy includes three-step chromatography (affinity – ion exchange – size exclusion).

HCD (host cell DNA) 

consists of DNA fragments derived from E. coli cells. Its biosafety risks include carcinogenicity, infectivity, and immunogenic reactions.

HCP (host cell proteins) 

are highly complex, with more than 2,000 proteins potentially present in an E. coli lysate. Their biosafety risks include protease-mediated degradation of therapeutic proteins and immunogenic reactions.

To ensure product safety, regulatory agencies have established strict limits on residual E. coli HCD and HCP levels.

Application Prospects of Biosynthetic Semaglutide

With the growing global focus on metabolic disease management, Semaglutide and other GLP-1 peptides continue to expand across multiple therapeutic areas:

Weight-loss therapeutics

Type 2 diabetes therapy

Combination metabolic treatment regimens

Development of next-generation GLP-1-based peptide drugs

The biosynthetic production model is poised to become the mainstream route for large-scale Semaglutide API manufacturing, offering high yield, purity, and cost efficiency.

 
Hzymes Biotech Peptide Product Portfolio Supporting Semaglutide Development

01.  Biosynthetic GLP-1 29 Peptide

Hzymes independently develops and manufactures GLP-1 (29 amino acids), expressed recombinantly in E. coli. Following enzymatic cleavage, chromatography purification, and lyophilization, a freeze-dried powder is obtained. Compared to chemical synthesis, this approach enables large-scale continuous production with high purity, low impurities, high yield, and reduced cost.

02. Recombinant Enterokinase (High Specificity)

Recombinant enterokinase (rEK) is produced via genetic engineering, offering high purity, activity, and specificity. It recognizes and cleaves proteins containing the Asp-Asp-Asp-Asp-Lys (DDDDK) motif.

Applications:

• Fusion protein processing in genetic engineering: removes N-terminal tags to ensure proper folding and functionality.
• Peptide drug manufacturing: processes insulin, its analogs, GLP-1, and others by removing N-terminal tags while preserving bioactivity.

03.  E. coli HCD Detection

Hzymes’ E. coli residual DNA detection kit adopts qPCR probe-based technology, combined with automated nucleic acid extraction systems for high-throughput applications. It provides accurate and efficient HCD detection solutions, suitable for quality control at various peptide production stages.

Hzymes recombinant enterokinase enables highly specific cleavage

04. E. coli HCP Detection

Enzyme-linked immunosorbent assay (ELISA) remains the most widely used method for HCP detection. The Chinese Pharmacopoeia recommends ELISA for host protein analysis, while the USP <1132> chapter also highlights ELISA as the preferred method.

Hzymes’ E. coli HCP kit uses a double-antibody sandwich one-step format, reducing detection time to just 2 hours, and enables rapid quantitative analysis of HCP content in samples.

Why Partner with Hzymes for Semaglutide Development

Hzymes provides a complete solution for Semaglutide and GLP-1 peptides:

Biosynthetic GLP-1 29 Peptide 

– high-purity, scalable, cost-effective production

Recombinant Enterokinase 

– precise N-terminal cleavage for active peptides

HCD & HCP Detection Kits 

– rapid, accurate quality control

Scalable Fermentation & Purification 

– continuous, pharmaceutical-grade manufacturing

Partnering with Hzymes ensures safe, efficient, and high-quality Semaglutide production.

Product Ordering

On October 10, 2025, BMS acquired in vivo CAR-T company Orbital Therapeutics for $1.5 billion;

On August 21, 2025, Gilead acquired in vivo CAR-T company Interius BioTherapeutics for $350 million;

On June 30, 2025, AbbVie acquired in vivo CAR-T company Capstan Therapeutics for $2.1 billion;

On March 17, 2025, AstraZeneca acquired EsoBiotec for $1 billion, entering the in vivo CAR-T field.

With continuous investments from major multinational corporations (MNCs), the in vivo CAR-T field has demonstrated its platform potential and market value.

Ex vivo CAR-T vs. In vivo CAR-T

CAR-T refers to a type of immunotherapy in which a patient’s own T cells are genetically engineered to express chimeric antigen receptors (CARs) that specifically recognize tumor-associated antigens on cancer cells, activating T-cell–mediated cytotoxicity.

The ex vivo CAR-T workflow involves:

cell collection and separation (obtaining leukocytes from peripheral blood and isolating T cells),

T-cell activation (using anti-CD3/CD28 antibodies),

viral transduction (delivering CAR genes via viral vectors),

in vitro expansion (culturing to amplify T cells),

and reinfusion (returning quality-controlled CAR-T cells to the patient).

However, this process is lengthy, costly, highly individualized, and shows limited efficacy against solid tumors.

The emerging in vivo CAR-T approach, in contrast, uses delivery vectors (such as LNPs or lentiviruses) to directly introduce CAR genes into endogenous T cells in vivo, effectively reprogramming them into CAR-T cells within the body. This eliminates steps like T-cell isolation, in vitro expansion, and lymphodepletion, reducing inter-patient variability and showing promise for solid tumor applications.

Ex vivo CAR-T vs In vivo CAR-T

Pathways to Achieve In vivo CAR-T: mRNA-LNP and Lentiviral Vectors

The key challenge of in vivo CAR-T lies in targeted delivery—how to precisely deliver CAR genes to T cells and achieve specific expression that activates their tumor-killing ability.

Currently, two main approaches are being explored: antibody-conjugated lipid nanoparticles (Ab-LNPs) and lentiviral vectors. These two platforms complement each other in terms of delivery mechanisms and expression characteristics, jointly advancing the development of in vivo CAR-T technology.

In vivo CAR-T Delivery Approaches

The mRNA-LNP Approach: A “Transient Expression System” for In Situ T-Cell Reprogramming

The mRNA-LNP approach uses antibody-conjugated LNPs (Ab-LNPs) to deliver CAR mRNA precisely into T cells in vivo, enabling in situ reprogramming.

Its core lies in Ab-LNP targeting modifications and multifunctional CAR molecular design, where antibodies bind to T-cell–specific surface receptors, overcoming the challenge of nonspecific delivery.

Preparation of Ab-LNPs and In Vivo CAR-T Expression

Preparation of Ab-LNP and In Vivo CAR-T Expression

01. Ab-LNP Targeting Mechanism: The “Molecular Bridge” for T-Cell Specificity

Through antibody modification, a specific “LNP–T-cell receptor” targeting mechanism is constructed:

Targeted enrichment: 

The surface of Ab-LNPs is conjugated with T-cell–specific antibodies (such as anti-CD3 or anti-CD8), which bind to receptors on circulating or splenic T cells and help avoid clearance by the liver’s reticuloendothelial system (RES).

Receptor-mediated endocytosis: 

The antibody–receptor interaction triggers endocytosis, internalizing Ab-LNPs into the T cell.

mRNA release: 

The ionizable lipid becomes protonated in the acidic endosomal environment, generating a proton sponge effect that disrupts the endosome and releases mRNA into the cytoplasm for efficient CAR protein expression.

02. Ab-LNP Structural Design: Synergistic Optimization of Four Components

Ab-LNPs rely on the coordinated design of antibodies, lipids, and helper components, all finely tuned for T-cell targeting and mRNA protection.

Surface Modification of LNP Determines Targeting Function

LNP surface modifications determine targeting functionality:

Targeting antibody: 

Should exhibit high specificity, low immunogenicity, and optimal affinity, typically conjugated via a PEG spacer arm. Common antibodies include anti-CD3 (pan-T-cell targeting), anti-CD8 (cytotoxic T-cell targeting), anti-CD28 (dual targeting and co-stimulation), and anti-Treg for regulatory T cells.

Ionizable lipid: 

pKa 6.2–6.5 (neutral in blood, protonated in endosomes); hydrophobic tails with mixed C14–C18 chains enhance T-cell membrane fusion; head groups with substituted amines (e.g., tetrahydroisoquinoline) improve π–π stacking with mRNA for stable complex formation.

Helper lipid and cholesterol: 

Helper lipids adjust membrane fluidity, while cholesterol stabilizes the nanoparticle structure. When the molar ratio of ionizable lipid:cholesterol ≥ 1:1, Ab-LNPs retain >90% encapsulation efficiency after 48 hours in plasma at 37 °C; reducing the ratio to 1:0.8 causes a sharp drop to 65%.

PEG-lipid: PEG2000-DMG 

forms a hydrated surface layer to extend circulation time and prevent the accelerated blood clearance (ABC) effect. Clinically, PEG-lipid content is typically 1–2% (molar) with biodegradable ester-linked PEG chains.

03. Key Process Parameters: The “Precision Scale” Determining Transduction Efficiency

The preparation of Ab-LNPs requires tight control of process parameters, as deviations can cause loss of targeting or increased toxicity.

04. Preclinical and Clinical Validation: From Animal Models to Human T-Cell Activation

Hematologic malignancies: 

In a joint study by the University of Pennsylvania and Capstan, CD8 antibody–modified Ab-LNPs delivering CD19 CAR mRNA achieved remarkable outcomes in leukemia mouse models:

1. Splenic CD8⁺ T-cell CAR expression rate: 68% (vs. 5.2% with unmodified LNPs);
2. 90% clearance of peripheral B cells (tumor-mimicking targets) within 24 hours;
3. In the high-dose group (2 mg/kg), complete tumor regression was observed in all three mice, with no liver toxicity.

Autoimmune diseases: 

In clinical trial HN2301, Prof. Zhu Chen’s team used CD8 antibody–modified Ab-LNPs with 22 antibodies per particle, ionizable lipid:mRNA ratio 10:1, and 96% encapsulation efficiency.

1. Among 5 patients with refractory lupus, high-dose infusion induced CAR expression in peripheral CD8⁺ T cells within 6 hours, sustaining B-cell clearance for 10 days.
2. After 3 months, the SLEDAI-2K score dropped from an average of 18 to 6 in 4 patients, with only mild (grade 1) CRS and no neurotoxicity.

Solid tumor studies: 

Academician Weihong Tan’s group developed dual-function Ab-LNPs co-delivering CAR mRNA and CCL21 mRNA.

1. In a melanoma mouse model, CCL21 guided CAR-T infiltration, increasing intratumoral CAR-T counts 3.8-fold compared with standard Ab-LNPs.
2. When combined with PD-1 inhibitors, tumor suppression reached 89% with no systemic toxicity, addressing the major challenge of poor CAR-T infiltration in solid tumors.

Product Information

Bibiliography

1.Wang Q, Xiao ZX, Zheng X, et al. In Vivo CD19 CAR T-Cell Therapy for Refractory Systemic Lupus Erythematosus. N Engl J Med. 2025;393(12):1125-1136.

2. Theresa L. Hunter et al. In vivo CAR T cell generation to treat cancer and autoimmune disease. Science 388, 1311-1317(2025). 

3. Bian, X, Guo, Q, Yau, LF. et al. Berberine-inspired ionizable lipid for self-structure stabilization and brain targeting delivery of nucleic acid therapeutics. Nat Commun 16, 2368 (2025). 

4. Davies M I, Cooper J D, Desmond S, et al. Advanced Drug Delivery Reviews, 2000, 45 (2-3): 169.

5. Carrasco, M.J, Alishetty, Alameh, MG. et al. Ionization and structural properties of mRNA lipid nanoparticles influence expression in intramuscular and intravascular administration. Commun Biol 4, 956 (2021). 

6. Zhang, X. D, Hou, L. L. In situ engineering of mRNA-CAR T cells using spleen-targeted ionizable lipid nanoparticles to eliminate cancer cells. Nano Today.

7. Nicolai CJ, Parker MH, Qin J, Tang W, et al. In vivo CAR T-cell generation in nonhuman primates using lentiviral vectors displaying a multidomain fusion ligand. Blood. 2024 Aug 29;144(9):977-987. 

8. Michels KR, Sheih A, Hernandez SA, et al. Preclinical proof of concept for VivoVec, a lentiviral-based platform for in vivo CAR T-cell engineering. J Immunother Cancer. 2023 Mar;11(3): e006292. 

9. Bauler M, Roberts JK, Wu CC, Fan B, Ferrara F, Yip BH, Diao S, Kim YI, Moore J, Zhou S, Wielgosz MM, Ryu B, Throm RE. Production of Lentiviral Vectors Using Suspension Cells Grown in Serum-free Media. Mol Ther Methods Clin Dev. 2019 Nov 26; 17: 58-68. 

10. Du L, Nai Y, Shen M, Li T, Huang J, Han X, Wang W, Pang D, Jin A. IL-21 Optimizes the CAR-T Cell Preparation Through Improving Lentivirus Mediated Transfection Efficiency of T Cells and Enhancing CAR-T Cell Cytotoxic Activities. Front Mol Biosci. 2021 Jun 4;8:675179.

11. Dai Q, Han P, Qi X, Li F, Li M, Fan L, Zhang H, Zhang X, Yang X. 4-1BB Signaling Boosts the Anti-Tumor Activity of CD28-Incorporated 2nd Generation Chimeric Antigen Receptor-Modified T Cells. Front Immunol. 2020 Nov 13; 11: 539654. 

12. Xu J, Liu L, Parone P, Xie W, Sun C, Chen Z, Zhang J, Li C, Hu Y, Mei H. In-vivo B-cell maturation antigen CAR T-cell therapy for relapsed or refractory multiple myeloma. Lancet. 2025 Jul 19;406(10500):228-231. 

13. Andorko JI, Russell RM, Schnepp BC, Grubaugh D, Mullen KF, Wakabayashi A, Carrington LJ, O'Malley T, Kuri-Cervantes L, Culp TD, Johnson PR. Targeted in vivo delivery of genetic medicines utilizing an engineered lentiviral vector platform results in CAR T and NK cell generation. Mol Ther. 2025 Oct 1;33(10):4937-4952. 

14. Jacob Garcia, Christine Dehner, Jeffrey Teoh, Wayne Wallis,A Phase 1, Multicenter, Open-Label Study of UB-VV111 in Combination with Rapamycin in Relapsed/Refractory CD19+ B-Cell Malignancies,Blood,Volume 144, Supplement 1, 2024, Page 1750.

15. Iscaro, Joshua, et al. Lyotropic liquid crystalline phase nanostructure and cholesterol enhance lipid nanoparticle mediated mRNA transfection in macrophages. Advanced Functional Materials 34.46 (2024): 2405286.

The Biochemical Role of Lactate Dehydrogenase (LDH)

Structure and Function

Lactate dehydrogenase (LDH) is an enzyme that catalyzes the interconversion of lactate and pyruvate with the concurrent interconversion of NADH and NAD+. This reaction is a crucial step in anaerobic glycolysis, a metabolic pathway that allows cells to produce energy under low oxygen conditions. The LDH enzyme exists as a tetramer, composed of four subunits, and is encoded by several genes, leading to the formation of different isoenzymes. The main isoenzymes are found in various tissues, such as:
 

– LDH-1 (H4): Predominantly in the heart.

– LDH-2 (H3M1): Found mainly in reticuloendothelial cells.

– LDH-3 (H2M2): Present in the lungs and other tissues.

– LDH-4 (H1M3): Found in the kidneys and pancreas.

– LDH-5 (M4): Primarily located in the liver and skeletal muscles.

Each isoenzyme has a slightly different affinity for lactate and pyruvate, reflecting its specialized role in different tissues. The balance between these forms of LDH is crucial for maintaining normal metabolic processes, especially in tissues with high energy demands.

Lactate Dehydrogenase (LDH)

Lactate Dehydrogenase (LDH), CAS: 9028-36-8, is a specific form of LDH that catalyzes the oxidation of D-lactate to pyruvate. While less commonly discussed than its counterpart L-Lactate Dehydrogenase, LDH plays a crucial role in certain metabolic pathways, particularly in microorganisms and some specialized human tissues. LDH is often utilized in research and diagnostic settings to study metabolic processes involving D-lactate.

Clinical Significance of LDH Levels

Tissue Distribution

LDH is widely distributed throughout the body, with significant concentrations in the heart, liver, kidneys, muscles (both skeletal and smooth), lungs, blood cells (both red and white), and brain. This widespread presence means that LDH is a useful marker for detecting tissue damage in a variety of organs.

LDH as a Biomarker

Due to its ubiquitous presence, elevated LDH levels in the blood can indicate a range of conditions where tissue damage has occurred. When cells are damaged, LDH is released into the bloodstream, making it a valuable marker for diagnosing and monitoring diseases such as:
 

– Heart attacks: 

Elevated LDH levels can indicate myocardial infarction, where heart muscle damage releases LDH into the blood.

– Liver disease: 

Conditions like hepatitis or cirrhosis can cause increased LDH levels due to liver cell damage.

– Muscle injuries: 

Trauma, strenuous exercise, or muscle diseases can lead to elevated LDH levels from muscle cell breakdown.

– Hemolytic anemia: 

The destruction of red blood cells releases LDH into the bloodstream.

– Cancer: 

Certain cancers, especially those with rapid cell turnover, can cause elevated LDH levels.

The specific pattern of LDH isoenzymes in the blood can sometimes help localize the source of tissue damage. For instance, a higher proportion of LDH-1 relative to other isoenzymes might suggest cardiac involvement, whereas elevated LDH-5 could indicate liver damage.

LDH in Medical Diagnostics

Lactate Dehydrogenase (LDH) is particularly relevant in the context of specific metabolic disorders, such as D-lactic acidosis. This condition can occur in individuals with short bowel syndrome or other gastrointestinal disorders where D-lactate is produced in excess by gut bacteria and accumulates in the body, leading to metabolic acidosis. Measuring LDH activity can help diagnose and manage this condition.
 

The LDH Blood Test: Purpose, Procedure, and Interpretation

Purpose of the LDH Blood Test

The LDH blood test is a diagnostic tool used to measure the level of LDH enzyme in the blood. This test serves several important purposes in medical practice:
 

– Detect tissue damage: 

Elevated LDH levels can indicate tissue damage from conditions such as heart attacks, liver disease, muscle injuries, or hemolytic anemia.

– Monitor disease progression: 

LDH levels can be used to track the progression of diseases like cancer, chronic kidney disease, or infections.

– Evaluate treatment effectiveness: 

In certain conditions, the LDH test can help assess how well treatment is working by monitoring changes in LDH levels over time.

Preparation and Procedure

Preparation: Generally, no special preparation is required before the LDH test. Patients are usually advised to continue with their normal activities and dietary habits. However, it is important to inform the healthcare provider about any medications or supplements being taken, as some substances can affect LDH levels.
 

Procedure: 

The LDH test is a simple procedure involving the collection of a blood sample. A healthcare professional will draw the sample from a vein, typically in the arm. The process is quick, usually taking only a few minutes, and is relatively painless.
 

Laboratory Analysis: 

Once the blood sample is collected, it is sent to a laboratory where the LDH level is measured using specific assays. The results are usually available within a few hours to a few days, depending on the laboratory.

Normal Range and Interpretation of Results

The normal range for LDH levels can vary slightly depending on the laboratory and the method used. Typically, normal LDH levels range from 140 to 280 units per liter (U/L) for adults. However, it is important to note that these values can differ based on factors such as age, gender, and overall health.

High LDH Levels: Elevated LDH levels may indicate tissue damage or disease. Conditions commonly associated with high LDH levels include:
 

– Heart attack (myocardial infarction)

– Liver diseases (e.g., hepatitis, cirrhosis)

– Muscle injuries or diseases

– Hemolytic anemia

– Certain cancers (e.g., lymphoma, leukemia)

– Infections (e.g., sepsis)

Low LDH Levels: Low LDH levels are less common but can occur in certain situations, such as:

– Genetic disorders affecting LDH production

– Chronic illness leading to reduced cellular turnover

Follow-Up: Elevated or abnormal LDH levels often require further diagnostic tests to pinpoint the exact cause of the tissue damage. This may include imaging studies, additional blood tests, or biopsies, depending on the suspected condition. It is important for patients to discuss their results with their healthcare provider to understand what they mean in the context of their overall health.

Hzymes Lactate Dehydrogenase (LDH) Products

For researchers and laboratories, LDH is available in various forms as a reagent, with Lactate Dehydrogenase being one of the key products. Hzymes offers a range of Lactate Dehydrogenase products under the following catalog numbers:

– HH1701-01
– HH1701-02
– HH1701-03
– HH1701-04
– HH1701-99

These products are typically used in research and diagnostic assays to measure LDH activity or to study metabolic pathways involving D-lactate. The availability of different catalog numbers allows researchers to choose the specific formulation or concentration that best suits their experimental needs.

Conclusion

Lactate dehydrogenase (LDH) is a vital enzyme involved in cellular respiration and energy production, playing a key role in the interconversion of lactate and pyruvate. Its widespread presence in various tissues makes it an important biomarker for detecting tissue damage and diagnosing a range of medical conditions, including heart attacks, liver disease, muscle injuries, and certain cancers. The measurement of LDH levels through blood tests is a valuable tool in clinical diagnostics, providing insights into the health and integrity of tissues.

Lactate Dehydrogenase (LDH), specifically, is an important enzyme in certain metabolic pathways and is particularly relevant in the diagnosis of conditions like D-lactic acidosis. In the research setting, LDH is available as a reagent from suppliers like Hzymes, supporting scientific studies in metabolism and enzymology.

As our understanding of LDH and its various isoenzymes continues to grow, so too does its importance in both clinical practice and research. Whether used as a diagnostic marker or a research tool, LDH remains a key enzyme in the study of cellular metabolism and disease.

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.

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Encapsulating mRNA in LNPs and then entering the human body not only has low toxicity, low immunogenicity, excellent kinetic stability, and robust structure, but also protects mRNA from nuclease destruction in the systemic circulation, and efficiently delivers mRNA into cells through a fusion mechanism with the lipid bilayer of early endosomes. It is the only mRNA delivery technology approved by the FDA and has been used in hundreds of millions of doses of mRNA vaccines around the world, and its safety and efficacy have been verified in this global new crown epidemic.

The development history of LNP technology

LNP type

According to the different structure and drug loading mechanism of lipid nanoparticles, LNPs can be divided into: cationic LNPs, Lipid polymer hybrid NPs (LPHNPs), calcium phosphate (CaP) LNPs (LCPs) and ionizable LNPs[1]。

01 Cationic lipid nanoparticles (Cationic LNPs).

Cationic LNPs were one of the first synthetic materials for gene delivery[2] and consist of cationic lipids, which have the ability to strongly bind and concentrate negatively charged nucleic acids into stable nanoparticles, known as LPX (lipoplexes), and neutral accessory lipids. This LPX is very effective in vitro but is unstable in aqueous solution and has limited efficiency in delivery in vivo due to their short half-life in the body circulation.

02 Lipid Polymer Hybrid NPsLPHNPs

LPHNPs are made up of nucleic acids concentrated with lipid and cationic polymers. This system combines the advantages of cationic lipids and polymers in gene delivery. Polycations reduce the amount of cationic lipids used and improve the nuclear delivery of DNA[3]. The lipid layer of the outer layer provides additional protection for nucleic acids and can be modified with different ligands and devices to improve pharmacokinetics and cell-specific targeting[1].

03 Lipid calcium phosphate NPs (LCP NPs).

LCP NP is the most versatile platform for the delivery of a wide range of nucleic acids. The nucleic acids combine with calcium phosphate (CaP) to form a core, which is then coated with different lipids to form a core-shell structure known as LCP-1. LCP nanoparticles are characterized by a small diameter, less than 50nm, which facilitates hepatocyte delivery through the sinusoidal window pores of the liver[4].

04 Ionizable LNPs

Ionizable lipid LNPs are typically made up of four components: ionizable lipids, phospholipids, cholesterol, and polyethylene glycol (PEG) lipids. Ionizable lipids are the most critical excipients in LNP delivery systems, and are particularly important in determining mRNA delivery and transfection efficiency, with ionizable cationic lipids having a pKa of less than 7 and therefore positively charged in acidic media and electrically neutral under physiological conditions. It can interact with nucleic acids and wrap them under acidic conditions (pH=4), and then change the buffer system to make pH=7.4 to form neutral LNPs, which are not only suitable for systemic drug delivery, but also avoid the disadvantages of permanently positively charged LNPs [5]; PEG lipids have a significant effect on the regulation of particle size and zeta potential, promote particle stabilization by reducing particle aggregation, and can also prevent serum proteins from binding to nanoparticles, reducing the clearance of nanoparticles in the systemic circulation, thereby prolonging the systemic circulation time. Phospholipids are generally neutral, providing bilayer structural stability for LNPs, while improving the permeability of cell membranes and promoting cell uptake and endosomal escape. Cholesterol improves particle stability by regulating membrane integrity and rigidity [6].

Ionizable lipid LNPs have two additional advantages, first, they bind to circulating endogenous proteins, such as apolipoprotein E (ApoE), which efficiently mediates the delivery of ionizable lipid LNPs to hepatocytes via the low-density lipoprotein (LDL) receptor pathway. Second, in the acidic pH environment of the endosomes, these lipids acquire a positive charge and can bind to the negatively charged lipids on the endosome membrane to form a non-bilayer structure, inducing the rupture of the endosomes, thereby causing the escape of the endosomes. Due to this potent endosomal escape process, the use of ionizable lipids for siRNA and mRNA delivery yields much greater gene silencing and expression in vitro than cationic lipids. These advantages have made ionizable lipids a key ingredient in some of the mRNA vaccines currently on the market, such as Moderna’s Covid-19 mRNA vaccine, which uses the self-developed ionizable lipid SM-102, and Pfizer and BioNTech licensed an ionizable lipid called ALC-0315 from Acuitas. In the following, we will focus on the preparation process of ionizable lipids.
 

Key quality parameters of LNPs

Key quality indicators during LNP production include particle size, polydispersity index (PDI), encapsulation efficiency, zeta potential, mRNA integrity of the formulation, and content of each component in the delivery system.

01 Particle size and PDI of LNPs

The average particle size and particle size distribution PDI of LNPs are important factors in the quality of LNPs and suitability for various applications, and are often characterized by dynamic light scattering (DLS). Studies have shown that LNPs with different particle sizes are expressed differently in vivo: the smaller the LNP size in the case of intramuscular administration, the more likely it is to migrate to the liver to show liver targeting, while the larger particle size is mostly left at the injection site [7]. In addition, the size of LNPs may affect the internalization, biodistribution, degradation, and clearance of LNPs, and different applications may require different particle sizes[8]. The optimal particle size range for LNPs is generally considered to be 20−200 nm with a PDI of less than 0.2.

02 Encapsulation rate of LNPs

Encapsulation efficiency is a very critical indicator of mRNA-LNP, which indicates the proportion of RNA encapsulated in lipid nanoparticles to total RNA, which is usually characterized by fluorescent dye method, which mainly affects the delivery efficiency, stability, and safety of drugs. High encapsulation efficiency LNPs can ensure that more drug molecules are effectively delivered to target cells, improving treatment efficacy; More stable in the in vitro and in vivo environment, resulting in less loss of the drug during delivery; It can also reduce the exposure of drug molecules, reducing the risk of immune reactions and side effects. Typically the default qualified LNP encapsulation rate is >90% [9].

03 The mRNA integrity of the LNP preparation

The stability and integrity of mRNA is crucial for the performance of its function, LNP, as the carrier of mRNA, encapsulates mRNA in it through specific technical means to protect mRNA from degradation by enzymes in the body, and at the same time helps mRNA to enter cells efficiently, if mRNA is degraded in LNP, it will affect its ability to code for proteins, and then affect the immune effect of vaccines. Therefore, the mRNA integrity parameters in LNPs are crucial in the quality control system, and the requirements for mRNA integrity of LNP preparations vary from industry to industry

04 Zeta potential of LNP

The LNP zeta potential, which is the electrostatic potential surrounding the LNP, is a good indicator of the stability of the nanoparticle suspension and its potential side effects, and can be measured by a variety of techniques such as electrophoretic light scattering (ELS), microelectrophoresis, and more. Zeta potential affects the colloidal stability, aggregation behavior, flocculation rate, and interaction with biofilms of LNPs, among other things. The higher the absolute value of the zeta potential, the higher the electrostatic repulsion and better stability of the LNP, while the neutral zeta potential leads to LNP agglomeration and potential agglomeration.

05 Identification and content determination of lipid components of LNP

The USP draft Procedures for the Quality Analysis of mRNA Vaccines states that the LNP lipid fraction and content of mRNA vaccines need to be determined. In order to meet the needs of method development and detection of mRNA-LNP, Hanhai New Enzyme established an applicable ELSD-HPLC analysis method, and the four LNP components were strongly retained, achieving complete separation and successful identification of lipid components.

A certain concentration of standard solution was injected into 10ul and 5ul respectively, a standard curve was established, and the unknown sample was diluted 10-fold and then injected for analysis, and the quantitative results were as follows:
 

Optimization idea of LNP preparation by microfluidic process

The commonly used preparation methods of lipid nanoparticles mainly include thin film hydration method, extrusion method, homogenization method, macrofluidics/T junctions, etc. However, the average particle size of LNPs produced by this method is relatively large, and the wrapping rate is low. At present, the most widely used is microfluidic hybrid technology, which is relatively simple and fast, mild and easy to scale-up.

The optimization of the preparation process of lipid nanoparticles is a complex process with multiple factors and parameters, which involves many aspects such as material selection, process parameter adjustment, and final product performance evaluation.
 

During the preparation of lipid nanoparticles, the molar ratio of each lipid component determines the composition of LNPs and affects their particle size, PDI, and efficacy. Due to the ability of PEG-lipids to inhibit the binding of ApoE to LNPs, excess PEG-lipids may adversely affect cellular uptake and transfection of LNPs. LNPs containing less PEG-lipids enable more efficient nucleic acid delivery. The two earliest marketed mRNA vaccines had a PEG-lipid ratio of 1.5% and 1.6%, respectively.
 

Studies have been shown [10][11] that the flow rate ratio of the aqueous and lipid phases and the total flow velocity in the aqueous and lipid phases significantly affect the particle size of LNPs. On this basis, Hanhai New Enzyme further studied the factors affecting the particle size of LNPs. The experimental results show that PEG%, TFR (Total flow velocity) and FRR (Flow rate ratio) are inversely proportional to particle size. The aqueous salt concentration is proportional to the particle size; N:P and aqueous pH have little effect on particle size.
 

Hzymes has explored and optimized the downstream purification process of LNP, mainly for the inlet flow rate, transmembrane pressure difference TMP (Trans-Membrane Pressure Drop), membrane pore size, membrane area, membrane material, hollow fiber and flat membrane cassette, the process time can be controlled at about 3.5h, the stability of mRNA-LNP preparations at -80°C can reach more than 18 months, and it can be frozen-thawed10 does not affect physical and chemical indicators.
 

Hzyme has advanced technology and rich experience in mRNA production and LNP preparation, and adopts simple, fast, high-stability, high-quality microfluidic technology for the preparation of mRNA-LNP, which can provide customized services for mRNA and mRNA-LNP with different specifications, and can customize LNPs with different particle sizes according to customer needs, and provide mRNA-LNP encapsulation efficiency, particle size, PDI, and Zeta potential and other related quality control testing services.

Based on the cumbersome preparation process of traditional LNPs, various equipment is required, resulting in high application costs and greatly limited throughput in early research or scientific research stages. In order to help customers quickly validate targets in the early research or scientific research stage, Hzymes Biotech

Hzyme provides:

A full set of mRNA vaccine & drug-related high-performance enzyme raw materials, chemical substrates, IVT kits and quality control kits, with independent plasmid preparation, IVT process development, mRNA purification, LNP encapsulation and related quality inspection platforms, can provide customers with a full set of solutions based on raw materials, comprehensive process development services, and help customers quickly promote the development of new drugs. For early-stage customers, we can provide CRO services, proof-of-concept services, third-party quality inspection services, and various finished mRNA stock solution products, including reporter genes, target proteins, gene editing proteins, and antigen proteins.

References

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[2] Malone RW, Felgner PL, Verma IM. Cationic liposome-mediated RNA transfection. Proc Natl Acad Sci U S A. 1989 Aug; 86(16):6077-81.
[3] Masuda T, Akita H, Harashima H. Evaluation of nuclear transfer and transcription of plasmid DNA condensed with protamine by microinjection: the use of a nuclear transfer score. FEBS Lett. 2005 Apr 11; 579(10):2143-8.
[4] Satterlee AB, Huang L. Current and Future Theranostic Applications of the Lipid-Calcium-Phosphate Nanoparticle Platform. Theranostics. 2016 Apr 27; 6(7):918-29.
[5] Tam YY, Chen S, Cullis PR. Advances in Lipid Nanoparticles for siRNA Delivery. Pharmaceutics. 2013 Sep 18; 5(3):498-507.
[6] Li X, Qi J, Wang J, Hu W, Zhou W, Wang Y, Li T. Nanoparticle technology for mRNA: Delivery strategy, clinical application and developmental landscape. Theranostics. 2024 Jan 1; 14(2):738-760.
[7] Di J, Du Z, Wu K, Jin S, Wang X, Li T, Xu Y. Biodistribution and Non-linear Gene Expression of mRNA LNPs Affected by Delivery Route and Particle Size. Pharm Res. 2022 Jan; 39(1):105-114.
[8] Eygeris Y, Gupta M, Kim J, Sahay G. Chemistry of Lipid Nanoparticles for RNA Delivery. Acc Chem Res. 2022 Jan 4; 55(1):2-12.
[9] Muramatsu H, Lam K, Bajusz C, Laczkó D, Karikó K, Schreiner P, Martin A, Lutwyche P, Heyes J, Pardi N. Lyophilization provides long-term stability for a lipid nanoparticle-formulated, nucleoside-modified mRNA vaccine. Mol Ther. 2022 May 4; 30(5):1941-1951.
[10] Roces CB, Lou G, Jain N, Abraham S, Thomas A, Halbert GW, Perrie Y. Manufacturing Considerations for the Development of Lipid Nanoparticles Using Microfluidics. Pharmaceutics. 2020 Nov 15; 12(11):1095.
[11] Malburet C, Leclercq L, Cotte JF, Thiebaud J, Bazin E, Garinot M, Cottet H. Taylor Dispersion Analysis to support lipid-nanoparticle formulations for mRNA vaccines. Gene Ther. 2023 May; 30(5):421-428.