The Magic of Enzyme Preservation – Decoding the Development of Enzyme Lyophilization Process

Challenges of Enzyme Lyophilization: How to Lock in 100% Activity

Biological products such as proteins, enzymes, and antibodies are prone to physical degradation (e.g., denaturation, aggregation) and chemical reactions (e.g., oxidation, hydrolysis) when in solution, leading to instability in their structure and function [1]. With the rapid development of biotechnology, an increasing number of biological products have begun to adopt lyophilization technology to extend their shelf life and improve transportation convenience. This method removes moisture from the substance by directly sublimating it from the solid state to the gaseous state, thereby preserving the original biological activity of the sample without damaging its structure, achieving long-term stable storage. However, during the actual lyophilization process, for biological active molecules such as enzymes that are extremely sensitive to environmental conditions, they may be affected by various adverse factors, including low-temperature stress [2], concentration effects [3], changes in pH value [4], phase separation [5], and dehydration stress, causing reversible or irreversible changes in the protein structure, thereby affecting activity. How to ensure the effective retention of enzyme activity before and after lyophilization has become one of the urgent technical problems to be solved. In addition, even under ideal storage conditions, enzyme preparations still face the risk of gradually losing activity during storage, which not only affects the final effect of the product but also increases production costs. Figure note: The wide application of lyophilization technology

Key Factors Affecting the Activity and Stability of Enzyme Lyophilization

Key Parameters of Lyophilization Process: Unlocking the Best Path

Lyophilization mainly includes key stages such as pre-freezing, annealing, primary drying, and secondary drying. Precise control of the conditions and parameters of each step is crucial for ensuring the biological activity and stability of enzyme preparations after lyophilization, among which the freezing temperature in the pre-freezing stage, the collapse temperature and heating rate in the primary drying stage, and the temperature and time in the secondary drying stage are particularly important.

• Key Point I of Lyophilization Parameters: Freezing Temperature

The freezing temperature determines the formation and size of ice crystals. An appropriate freezing temperature can form fine and uniform ice crystals, which helps to improve sublimation efficiency and reduce damage to the structure of biological molecules. The freezing temperature should be 10-20℃ lower than the eutectic point to ensure that all solutes are completely solidified. James A et al. [6] improved the non-uniformity of sample nucleation by controlling the nucleation temperature and rate, which not only enhanced the stability of the sample but also increased the initial drying efficiency by 3.5 times.

• Key Point II of Lyophilization Parameters: Collapse Temperature and Heating Rate in the Primary Drying Stage

The collapse temperature is the temperature at which the rigidity of the solid matrix is insufficient to maintain the honeycomb structure when the temperature of the drying layer reaches a certain critical value, leading to the collapse of the cavity wall. Controlling the drying temperature below the collapse temperature can prevent structural collapse and ensure the smooth progress of the sublimation process. Differential scanning calorimetry (DSC) or other methods can be used to determine the collapse temperature, and the temperature during the primary drying stage should be kept below this temperature. The following figure shows the internal microscopic structure of human serum albumin during the lyophilization process, gradually collapsing due to temperature rise [7].

Figure note: Microscopic cross-sectional view of human serum albumin during the lyophilization process

A: Sample freezing at the air/liquid interface (1*), B: Drying interface advancing along the front (2*), C: Temperature rise, front collapse (3*) and surface rupture (4*)

The heating rate affects the sublimation speed and uniformity of moisture. Too fast or uneven heating can cause local overheating, thereby destroying the sample structure. Slow and uniform heating helps moisture to sublimate uniformly and avoids local overheating.

● Key Point III of Lyophilization Parameters: Temperature and Time in the Secondary Drying Stage

The secondary drying stage is used to remove residual bound water and thoroughly dry the sample. Setting an appropriate temperature (higher than the primary drying temperature but lower than the sample’s tolerance temperature) and sufficient time to completely remove residual moisture can ensure that the sample is completely dried, while avoiding excessive drying that leads to sample denaturation.

The Magic of Formulation: How Do Lyophilization Protectants Play a Role in the Lyophilization Process

Lyophilization protectants play multiple roles in the lyophilization process. They can prevent physical damage and chemical degradation, maintain the stability and activity of biological products (such as enzymes, proteins, and antibodies), and enhance the reconstitution ability of the sample. The selection principles of lyophilization protectants usually include glass transition temperature (Tg), water displacement ability, the ability to maintain structural stability, and the ability to enhance reconstitution, in addition to considering antioxidant ability, chemical stability, biocompatibility, and cost-effectiveness according to specific application scenarios.


Among them, Tg refers to the temperature at which the protectant transitions from the rubbery state to the glassy state, playing a crucial role in the stability of proteins after lyophilization. Selecting protectants with a higher Tg, such as trehalose and sucrose, can ensure that the sample remains in the glassy state during the primary drying stage, preventing protein denaturation and degradation during storage, thereby maintaining structural integrity.


Sugars and polyhydroxy compounds are the most common and widely used class of lyophilization protectants, among which disaccharides are the most effective protectants, especially sucrose and trehalose [9], due to their high glass transition temperature (Tg) and good water displacement ability and structural stability. Jovanović Natasa et al. [10] observed the particle morphology of myoglobin and lysozyme during the lyophilization process through scanning electron microscopy and found that the particle morphology was affected by lyophilization stress without the addition of protectants, while sucrose and trehalose could effectively protect the particle morphology from being affected.


By making microscopic changes to maintain the activity and stability of enzymes during the lyophilization process, Kawai Kiyoshi et al. [11] used four disaccharides as lyophilization protectants for lactate dehydrogenase, effectively reducing the activity loss of lactate dehydrogenase during the lyophilization process and enhancing its stability during long-term storage.

Development Ideas for Enzyme Lyophilization Process

The development of the lyophilization process is a systematic and complex process, involving multiple steps such as eutectic point testing, collapse temperature testing, sublimation rate testing, and lyophilization protectant screening. It is necessary to comprehensively analyze key influencing factors and conduct systematic experimental design to obtain a good lyophilization process.


In the development of a biochemical diagnostic enzyme raw material by Hzymes, due to the low collapse temperature of the solute and sample mixture and the low concentration of the mixture, the product after lyophilization appeared to be surface collapsed, non-uniformly dispersed, and unable to form a good lyophilized powder cake shape; more troublesome is that after lyophilization with only buffer components, the enzyme activity and the 37℃ heat stability incubation process of the lyophilized powder both decreased significantly (Formula 1.0).

The problem of product collapse is mainly due to the high primary drying temperature, so it can be solved by lowering the drying temperature. However, to form a honeycomb structure and maintain the stability of the enzyme, it is necessary to carry out the screening and combination of protectants. In the preliminary screening, by evaluating their protective effects on enzyme activity, the advantageous lyophilization protectants were obtained: Formula 1.5 and Formula 1.6; further combination and concentration optimization of Formula 1.5 and 1.6 were carried out, and after 3 rounds of testing, the best formula with high lyophilization activity and good heat stability was finally obtained: Formula 3.4. At the same time, through the optimization of primary drying temperature → protectant screening → lyophilization buffer solution formula, a good lyophilized appearance was finally obtained.

Step 1: Due to the high primary drying temperature exceeding the collapse temperature, the phenomenon of product collapse occurred.

Step 2: Optimize the drying temperature to solve the collapse problem, but the product appears to be non-uniformly dispersed, mainly because the lyophilization structure is not rigid enough to form a good honey comb structure.

Step 3: Through the screening of protectants, the lyophilization morphology was improved, but due to the high surface tension between the sample and the vial, the product climbing phenomenon occurred.

Step 4: Adjust the lyophilization formula to improve the solution surface tension and obtain a good lyophilized product appearance.

Hzymes Lyophilized Products and Lyophilization Development Platform

PCR Lyophilized Reagents

PCR lyophilized reagents refer to the lyophilization treatment of various components required for PCR reactions (including DNA polymerase, reverse transcriptase, dNTPs, MgCl₂, buffer, and primers, etc.), preparing them into stable powder or lyophilized ball products. After reconstitution with water, PCR reactions can be carried out directly. Compared with traditional liquid PCR reagents, lyophilized reagents have strong stability, high portability and ease of use, and can be adapted to automated equipment, greatly expanding the application range of PCR technology in real scenarios such as pathogen rapid diagnosis, gene detection, and forensic identification.

Hzymes provides MS qPCR/RT-qPCR lyophilized premix, supports the addition of primers and probes for lyophilization, and can be used directly after adding sample nucleic acids; supports singleplex/multiplex detection; uses the UDG-dUTP anti-contamination system, which can effectively prevent the risk of aerosol contamination of amplification products.

qPCR Premix Lyophilized Ball Product Display

LAMP Lyophilized Reagents and Customization Services

LAMP lyophilized reagents refer to the lyophilization treatment of various components required for LAMP reactions (including Bst enzyme, reverse transcriptase, dNTPs, MgCl₂, buffer, and primers, etc.), preparing them into stable powder or lyophilized ball products. After reconstitution with water, LAMP amplification reactions can be carried out directly. Compared with traditional liquid LAMP reagents, lyophilized reagents have strong stability, are easy to use, and can be stored at room temperature, greatly reducing the cost of cold chain transportation.

Hzymes provides LAMP/RT-LAMP lyophilized premix and products, and also provides customized CRO/CDMO lyophilization services, meeting the diverse needs of customers and customizing special lyophilized products for customers.

Lyophilized Calibration Quality Control Products

Hzymes innovatively uses animal-derived blood matrix to make glycated hemoglobin calibration quality control products, which are cost-effective and easy to obtain, suitable for mass production, and conducive to industrial production; users can order one year or more at a time, avoiding frequent changes in target values and control charts. While increasing the production of glycated hemoglobin calibration quality control products, it effectively avoids biological safety issues and import and export regulatory risks.

Lyophilized CMO Services

As a leader in the specialty enzyme industry, Hzymes has rich experience in the development and production of recombinant protein products, is good at E. coli and yeast expression systems, and has built a super clean enzyme purification platform and rich lyophilized formulation development technology through long-term project research and development, core technology accumulation, and industrialization in-depth cultivation. It can provide lyophilization platforms of different scales such as 0.4m², 0.5m², 2m², 10m², and 20m², which can match the production needs of small-scale trials, medium-scale trials, and industrialization.