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Free Thiol Quantification

Free thiols are a unique structure formed by the sulfur atom in cysteine. Free thiols are very reactive and can bind with other proteins to alter their structure and function, or react with oxygen molecules to produce oxides that damage cells and tissues. Quantifying free thiols is an important test for biological products such as antibodies. For example, the biological function of antibodies containing free thiols can sometimes be lower than that of complete antibodies, sometimes substantially so. Therefore, rapid response and accurate quantification of free thiols in protein-based biological products is crucial for early process development and later production monitoring.

Thiols and disulfide bonds often participate in enzyme catalysis, cofactor binding, and maintenance of protein active conformations as active groups. The content of free thiols is highly related to the properties and existence of proteins and is an essential part of quality testing in the biopharmaceutical process.

The Ellman method is a conventional method for detecting free thiol concentration. The principle involves the reaction of DTNB reagent with free thiols to produce 2-nitro-5-thiobenzoic acid (TNB-), which can ionize in water under neutral or alkaline pH conditions to generate TNB2-divalent anions. This TNB2-ion appears yellow, and its concentration can be quantified by measuring absorbance at 412 nm visible light.2-Then, using cysteine standards or molar absorptivity-based methods, free thiols can be quantified.

BTP Biotech has established seven testing platforms under the dual quality certification system of CNAS/ISO9001, including the free thiol content testing platform, which is CNAS certified and has comprehensive methodological development and transfer capabilities. BTP also provides otherbiological product characterizationrelated services, which can be clicked to learn more.

BTP Biotech Biological Product Characterization Service Content