Quantification of Free Sulfhydryl Groups in Recombinant Protein Vaccines

Recombinant protein vaccines are prepared using genetic engineering technology, with the aim of eliciting an immune response by expressing and producing specific proteins of pathogens. These vaccines do not contain complete active pathogens but select specific proteins or parts of the pathogens as antigens. In the structure of proteins, mercapto mainly exists in cysteine, playing a key role in protein folding and stability. By forming disulfide bonds, it helps maintain the three-dimensional structure of proteins. When mercapto is free and does not form disulfide bonds, it is called free mercapto.

 

Quantitative detection of free mercapto is an important analytical method used to detect the oxidation state and stability of proteins. In the manufacture process of recombinant protein vaccines, excessive free mercapto may cause protein structure instability, affecting the quality and effectiveness of the vaccine. Therefore, quantitatively analyzing the free mercapto in the vaccine through scientific methods is crucial for ensuring the quality and safety of the vaccine.

 

Current commonly used methods for quantifying free mercapto mainly include DTNB, fluorescence, and mass spectrometry. DTNB (5,5'-dinitro-2,2'-dithiodibenzoic acid) is a common chemical reagent used to determine free mercapto. This reagent reacts with free mercapto to produce a yellow product, the absorbance of which can be measured at 412nm, thus quantifying the amount of free mercapto. Its operation is simple, but its sensitivity is relatively low, which may not be able to detect low content of free mercapto; it may also be interfered by other reducing agents such as DTT, β-mercaptoethanol, etc. The fluorescence method usually uses a fluorescent probe that can react with mercapto and emit fluorescence, and by measuring the fluorescence intensity, the amount of free mercapto can be quantified.

 

Mass spectrometry can be used to accurately quantify the amount of free mercapto in recombinant proteins. In this method, the protein is first treated to protect the free mercapto, then labeled with a reagent that can react with mercapto (such as hydroxylamine), and finally quantified by mass spectrometry. Mass spectrometry has high sensitivity, can detect very low concentrations of free mercapto, and provides accurate location information of free mercapto in recombinant proteins, making it the preferred method for analyzing complex samples.

 

MtoZ Biolabs provides drug quality research services in compliance with global pharmaceutical regulations for customers. We have developed a mass spectrometry-based recombinant protein vaccine free mercapto quantification technology with our powerful bioanalytical capability. This technology allows researchers to accurately determine the amount of free mercapto in recombinant protein vaccines, which is crucial to ensure the function and stability of recombinant protein vaccines. Additionally, this technology can also be used to detect whether the mercapto in the recombinant protein vaccine has been oxidized during expression and purification, and whether incorrect pairing of mercapto has occurred. MtoZ Biolabs can also provide customized free mercapto quantification technology services according to customers' specific needs. Free consultation is welcomed.