Objective: To establish a fluorometric method for the determination of trace amount free sulfhydryl groups in recombinant protein drugs. Methods: The Measure-iTTM Thoil Assay Kit based on fluoresence was used to detect free sulfhydryl groups. First, the protein denaturation conditions (urea, guanidine hydrochloride) were explored and optimized to determine the types and conditions of denaturation agents. The optimized method was verified by recombinant protein drugs. The results met the verification standards. However, in the testing of PEG-GCSF and KGF (molecular theory contains 1 mol free sulfhydryl/mol protein) products by the above method, it was found that the detection value of free sulfhydryl group was significantly lower than the theoretical value. The highest detection rate was only 29% of theoretical value, which may be related to the insufficient exposure of free sulfhydryl group inside protein molecule. In this study, free thiol in recombinant protein drug molecules was fully exposed by protease enzyme creative way. Other key parameters were optimized, and the methodology verification of the optimized analysis method was completed. Results: By screening the denaturation conditions of the samples before treatment, the optimal pre-treatment conditions were using 8 mol·L-1 urea to incubate for 30 min at 37 ℃. To solve the problem that sulphur content of recombinant protein 1 and recombinant protein 2 was much lower than the theoretical value, PEG-GCSF and KGF were first treated by proteases cut at the optimal protease conditions. Proteinase K [protein: protease K (20∶1,w∶w)], 37 ℃ incubation for 1 h. The measured values after enzymolysis were consistent with the theoretical values. It has been verified that the precision of the results is less than 15%. The recovery rate of each concentration is in the range of 80%-120%. The linear range of free sulfhydryl group was 1-15 μmol·L-1 (r>0.99). The limit of quantification (LOQ) was 1 μmol·L-1. All the verification indicators are in line with the verification standards and met testing requirements. Conclusion: In this study, protease digestion was used for pre-treatment of samples to fully expose free sulfhydryl groups in protein drug molecules. It provides a method and reference for quantitative determination and characterization of trace free sulfhydryl groups in this kind of protein molecules.
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