Background

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.

Development process of freeze-dried products

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.

Figure note: The impact of lyophilization protectants on enzyme activity

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.

Product Overview

This product contains reaction buffer, RT enzymes mix of Bst DNA polymerase and heat-resistant reverse transcriptase, lyophilized protectant and fluorescent dye components. The reaction buffer contains Mg ²⁺ , dNTP and other necessary components for amplification, users can directly mix the reaction buffer, RT enzymes mix, fluorescent dye, primer, and then add the template. If necessary, users can add the lyophilized protectant to prepare lyophilizable system.

Performance Data

High sensitivity and high inhibitor tolerance

RT-LAMP reaction is performed using a nasal swab sample spiked with SARS-CoV-2 pseudovirus (50 copies/T, 100 copies/T, and 250 copies/T) after a simple treatment with Hzymes Sample release buffer (HMD3504).

High specificity

There is no NTC amplification while using RT-LAMP  fluorescent master mix (Liquid, lyophilization ready)  (HMD5207).

Performance consistency with lyophilized reagent

dUTP tolerance

Transportation and storage conditions

Transported with dry ice, stored at -25～-15℃. Avoid repeated freezing and thawing, the product is valid for 12 months.