Molecular diagnostic technology distinguishes itself from various detection methods due to its exceptional sensitivity and specificity. However, conventional liquid diagnostic reagents suffer from drawbacks like high transportation expenses and a limited shelf life, which constrain their utility. In response to evolving market needs and technological advancements, lyophilized molecular diagnostic testing reagents have emerged. Freeze-dried products offer several advantages over traditional liquid testing reagents, including:

• Stable at room temperature - the three-dimensional structure is fixed, which can ensure the stability of the enzyme and reaction system at room temperature. No need for cold chain transportation and storage in refrigerators or cold storage, which can extend the shelf life of products.
• Easy to use- No need for repeated freezing and thawing, liquid dispensing, etc.
• Reduced cost- No need for cold chain transportation and frozen storage, effectively reducing transportation and storage costs.
• High precision- One freeze-dried microcore is used in one reaction to reduce the composition error during reagent preparation, shorten the operation time, prevent the loss of biological activity during liquid preparation, and fundamentally improve the precision.
• Flexible use- Not limited by sample number, one sample can also be loaded.

Conventional molecular enzymes or premixes frequently incorporate high concentrations of glycerin as a cryoprotective agent and stabilizer. However, the presence of glycerin in the reagent can disrupt the water's structural integrity, making it challenging to eliminate water effectively. This, in turn, has a detrimental impact on the structure and stability of the freeze-dried product. Therefore, when opting for freeze-drying, it is advisable to select glycerol-free reagents.

Furthermore, in applications that are particularly sensitive to reagent composition, such as digital PCR, the utilization of high-concentration glycerin-free enzymes can further mitigate the influence of glycerol on the digital PCR reaction. This approach not only minimizes interference but also broadens the potential for optimizing the digital PCR detection reagent system.

YEASEN Glycerol-free molecular enzyme solution

YEASEN has established a comprehensive non-glycerin molecular enzyme R&D and production platform, and has achieved large-scale production of multiple Glycerol-free molecular enzymes through material selection, environmental control, process optimization, and quality assurance.

• Trial of dialysis process

Optimizing the dialysis process for a fixed volume of glycerin enzyme:

1. Screening for the optimal dialysis buffer for different molecular enzymes;
2. Screening for stable storage solutions for different molecular enzymes to allow long-term storage at 4℃ or -20℃.

• Tangential flow filtration process amplification

Based on the dialysis process screening, the optimal dialysis buffer components and their optimal storage solutions can be determined. Subsequently, tangential flow filtration process (also known as UFDF) can be used for solution buffer replacement.

 

Figure 1: Schematic diagram of tangential flow filtration system

The crucial factors in the development of the tangential flow filtration process revolve around the choice of storage solution formula, washing and filtration solution formula, as well as the transmembrane pressure and flow rate applied during filtration. YEASEN has established a standardized approach for tangential flow filtration, enabling the swift determination of washing and filtration volumes and time parameters based on the desired glycerin content in the target molecular enzyme. This approach helps in economizing on both materials and time.

 

Figure 2: Experimental flow of tangential flow filtration system

• Quality control

The various preparation processes outlined above are designed to accommodate the diverse requirements for different volumes and product types. To assess product stability across various batches and within the same process, YEASEN has developed a comprehensive performance parameter characterization system. This system allows for a thorough evaluation of the impact of glycerol-free measures from multiple perspectives. Characterization methods encompass protein quantification, enzyme activity assessment (recovery rate), glycerin content analysis, protective agent content assessment, and enzyme performance evaluation, among others.

YEASEN glycerol-free molecular enzyme products

YEASEN has adopted a glycerol-free molecular enzyme preparation method to produce enzymes without glycerin content from those initially containing glycerin. Presently, we can supply substantial quantities of glycerol-free enzyme raw materials suitable for qPCR/RT-qPCR and LAMP/RT-LAMP applications. These raw materials are designed for freeze-drying in diagnostic testing, all the while maintaining the same level of quality and performance as their liquid counterparts.

Glycerol-free molecular enzyme products

qPCR premixes were prepared using YEASEN 14316ES and corresponding glycerol-containing enzymes, respectively, and the template of 10⁵, 10⁴ and 10³ copies/mL were amplified at the same time. The results showed that the amplification effect of glycerol-free 14316ES was consistent with that of glycerin-containing counterpart. The accelerated stability test of 13705ES was performed at 37℃. The results showed that the expansion performance of 13705ES was consistent with that of unaccelerated control after 54 days of accelerated treatment at 37℃. The above results showed that there was no difference in the performance of glycerol-free enzyme and glycerin-containing counterpart, and the stability was good.

 

Figure 3: Glycerol-free enzyme product test data

• RT-qPCR freeze-dried products: Accelerated at 50℃ for 25 days, freeze-dried products have stable morphology and performance

Yeasen 13705ES was prepared into RT-qPCR premixed solution, which was freeze-dried and tested for accelerated stability at 50℃ for 25 days. At the same time, 10⁵, 10⁴ and 10³ copies/mL of the novel coronavirus pseudovirus template were amplified. The results showed that the product morphology, Ct value and fluorescence value had no significant change before and after freeze-drying, and the freeze-drying product had good stability.

  

Figure 4: Accelerated stability test data of RT-qPCR freeze-dried products

• RT-LAMP freeze-dried products: Accelerated at 45℃ for 30 days, freeze-dried products have stable morphology and performance

The RT-LAMP premix was prepared with YEASEN 14405ES and 11297ES, and the performance test was carried out after freeze-drying. The positive reaction hole was orange and the negative reaction hole was magenta, which showed great contrast, and the result was clearly interpreted. In addition, the morphology of freeze-dried products did not change significantly after 30 days of acceleration at 45℃. The results showed that the freeze-dried product was stable in shape and performance.

 

 

Figure 5: RT-LAMP freeze-dried product test data

 

Product information

Classification	Name	Catalog No.
Glycerol-free enzyme products for qPCR/RT-qPCR	Hieff Unicon® V Lyo-nCoV Multiplex One-Step RT-qPCR Kit	13775ES
	Hieff UNICON® HotStart E-Taq DNA Polymerase, Glycerol-free	14316ES
	Hifair® V Reverse Transcriptase, Glycero-free	11301ES
	Uracil DNA Glycosylase (UDG), Glycerol-Free	10707ES
	Murine RNase Inhibitor (200 U/μL, Glycerol-free)	10703ES
Glycerol-free enzyme products for RT-LAMP	Hifair® III Reverse Transcriptase, 600U/μl, Glycerol-free	11297ES
	Lyophilized Bst Plus DNA Polymerase (60 U/μL，Glycerol-Free)	14405ES