Development and verification of a reversed-phase high-performance liquid chromatography method for determination of protein content in recombinant human thymosin beta 4 eye drops
10.13200/j.cnki.cjb.004401
- VernacularTitle:重组人胸腺素β4滴眼液蛋白含量反相高效液相色谱检测方法的建立及验证
- Author:
ZHANG Bo
- Publication Type:Journal Article
- Keywords:
Reversed-phase high-performance liquid chromatography(RP-HPLC);
Recombinant human thymosin beta 4 eye drops;
Protein content
- From:
Chinese Journal of Biologicals
2025;38(1):96-101
- CountryChina
- Language:Chinese
-
Abstract:
Objective To develop and verify a reversed-phase high-performance liquid chromatography(RP-HPLC) method for the determination of protein content in recombinant human thymosin beta 4(rh-Tβ4) eye drops for the quality control of protein content in rh-Tβ4 eye drops.Methods A ZORBAX 300SB-C18(4. 6 mm × 250 mm, 5 μm) chromatographic column was used for gradient elution with ultrapure water, acetonitrile and trifluoroacetic acid as mobile phases. The UV detection wavelength was 210 nm, the injection volume was 100 μL, the flow rate was 1. 0 mL/min, the column temperature was 25 ℃,and the sample room temperature was 5 ℃. The protein content in the eye drops was calculated by standard curve method.The system applicability, specificity, linearity and range, accuracy, repeatability, intermediate precision, solution stability and durability of the method were verified. The protein contents of four batches of rh-Tβ4 eye drops was detected by using the developing RP-HPLC.Results The theoretical number of trays for the main peak of system applicability was 192 039,185 819 and 186 348 respectively, all of which were not less than 5 000. The separation degree between the main peak and adjacent peaks was 3. 88, 3. 79 and 3. 76 respectively, all greater than 1. 5, indicating that the system applicability was qualified. The blank solvent and blank control did not affect the main peak integration of recombinant rh-Tβ4, and did not interfere with the detection results, indicating that the specificity was good. The standard curve for protein content within the range of 50 μg/mL to 150 μg/mL showed good linearity, with the correlation coefficient(R~2) of 0. 992 3, greater than 0. 99.The recovery rates of high, medium and low concentration standards were 98. 01%, 99. 17% and 97. 70% respectively. The RSD of protein content in 6 samples with the same concentration was 1. 38%. The RSD of protein content in the same sample detected by different experimenters on different dates was 1. 05%. Within 0, 6, 12, 18 and 24 h, the RSD of protein content in the same test sample was 0. 36%. The RSD of protein content of the test sample was 0. 45% at the column temperature of 23 ℃, 25 ℃ and 27 ℃. In addition, the protein contents of four batches of rh-Tβ4 eye drops were 0. 51, 0. 51, 0. 49 and0. 50 mg/mL respectively, which were consistent with the labeled amount.Conclusion A RP-HPLC method for the determination of protein content in rh-Tβ4 eye drops has been developed, which was proved to be accurate and reliable, and can be used for the quality control of protein content in rh-Tβ4 eye drops.
