Development and verification of a modified Lowry method for determining the protein content of recombinant respiratory syncytial virus-like particle vaccine
10.13200/j.cnki.cjb.004737
- VernacularTitle:用于检测重组呼吸道合胞病毒病毒样颗粒疫苗蛋白含量改良Lowry法的建立及验证
- Author:
Wenxi HUANG
- Publication Type:Journal Article
- Keywords:
Respiratory syncytial virus(RSV);
Virus-like particle(VLP) vaccine;
Protein content;
Lowry method
- From:
Chinese Journal of Biologicals
2026;39(07):846-851+859
- CountryChina
- Language:Chinese
-
Abstract:
ObjectiveTo develop a modified Lowry method for determining the protein content of recombinant respiratory syncytial virus(RSV)-like particle vaccine, optimize and verify the method, so as to apply it to the accurate detection of protein content of the vaccine.MethodsThe polysorbate 20 in the vaccine was hydrolyzed with sodium hydroxide(NaOH),and sucrose small molecule interferents were removed by thermal precipitation with trichloroacetic acid(TCA), to develop a modified Lowry method for protein content detection. The NaOH concentration(3 and 4 mol/L), centrifugation time(10 and30 min), TCA concentration(35%, 40% and 45%) of the method were optimized, and the specificity, linear range, accuracy,precision and dilution reliability of the method were verified. The developed method was used to detect the protein content of five batches of recombinant RSV-like particle vaccine drug substances, the corresponding final bulk products and freeze-dried finished products.ResultsThe optimal conditions were determined to be NaOH concentration of 4 mol/L, centrifugation time of 30 min, and TCA concentration of 40%. The bovine serum albumin(BSA) national standard showed a good linear relationship with A_(750)in the final concentration range of 0-120 ??g/mL, and the linear regression equation was Y = 0. 004 354 X +0. 074 28, R~2= 0. 997. This method could effectively eliminate the interference of polysorbate 20 and sucrose in the sample buffer. The recovery rates of accuracy verification were within the range of 90%-100%, and the coefficients of variation(CVs) of repeatability and intermediate precision verification were both less than 10. 0%. The recovery rates of the test results of vaccine bulk solution at different dilution factors were all within 85%-115%, with the CVs < 5. 0%. The protein content of five batches of vaccine bulk solutions was 1. 34-1. 57 mg/mL, and the protein content of corresponding final bulk and freeze-dried finished products was 112-133 ??g/mL.ConclusionThe modified Lowry method has good specificity, accuracy, precision and dilution reliability, and is suitable for the detection of protein content in recombinant RSV-like particle vaccine.
