Freeze-drying, also known as lyophilization, is a process where materials are sublimated under frozen conditions. Throughout this process, the materials undergo stages of low temperature, freezing, and vacuum sublimation. Initially, water in the solution crystallizes into ice, while the remaining water and solutes gradually form a highly concentrated liquid, ultimately freezing together. These changes impose various stresses on enzymes within the system. Firstly, there's the stress of low temperatures. Additionally, the formation of the highly concentrated liquid can lead to changes in ion strength and pH, further pressurizing the enzymes. Finally, dehydration also poses a stress, potentially causing protein precipitation or inactivation. Hence, to maintain the activity of biomacromolecules during freeze-drying, it's essential to add various protective agents to overcome these stresses. Although many studies have summarized this from different angles, such as the role of protective agents and types of compounds, applying these principles to molecular diagnostics presents its own challenges.

On one hand, the freeze-drying process for molecular diagnostics demands higher standards. It relies on enzyme activity for amplification and subsequent detection, requiring not only intact protein sequences but also preserved spatial structures. On the other hand, molecular diagnostics itself adheres to specific rules. Directly applying the principles of drug freeze-drying to molecular diagnostics may lead to complications. For instance, various amino acids commonly used in drug freeze-drying often interfere with molecular diagnostics. Therefore, freeze-drying for molecular diagnostics has its own set of principles. Combining molecular diagnostics, we can briefly elucidate the principles of freeze-drying agent development.

Principle One: Utilize molecular biology-grade materials

This is an often-overlooked issue. We are accustomed to using reagents (suppliers) that have been screened for "molecular biology-grade" during routine development. However, in the development of freeze-drying protective agents, the quality standards of reagents are crucial. If certain reagents do not meet the requirements, it may affect the performance of freeze-dried reagents. Therefore, ensuring the quality of raw materials is crucial.

Principle Two: Prioritize the freeze-drying process

Consideration of the freeze-drying process is crucial. Firstly, pay attention to the change in volume after freeze-drying. Some customers only discover that the volume after freeze-drying exceeds expectations after determining the system and plate, leading to subsequent issues. Additionally, pay attention to the preservation of enzyme activity. Although in most cases there is no significant difference in enzyme activity before and after freeze-drying, in reality, it is challenging to completely preserve enzyme activity. Therefore, it is necessary to prioritize the effects of freeze-drying during development to fully leverage the advantages of freeze-dried reagents.

Principle Three: Avoid using volatile reagents

During the freeze-drying process, it is advisable to avoid using volatile reagents such as DMSO, TMAC, and formamide. These reagents may evaporate during the sublimation process, affecting the freeze-drying results. It is more ideal to choose formulas without glycerol or with extremely low concentrations of glycerol.

In summary, the development of freeze-drying protective agents requires consideration of various factors, especially in the field of molecular diagnostics. By adhering to the above principles, the performance and stability of freeze-dried reagents can be effectively improved, thus better applied in experiments and diagnostics. At SBS Genetech, we offer a range of freeze-dried enzyme raw materials and finished microbeads covering PCR, LAMP, RPA, CRISPR, and other systems, bringing more progress to researchers.