RESUMO
@#Objective To develop and verify a whole column imaging detection-capillary isoelectric focusing(WCID-CIEF)method for the determination of isoelectric point(pI)of pertactin(PRN).Methods The WCID-CIEF method for the determination of PRN antigen was developed by optimizing the parameters such as the focusing time and final concentration of samples in the WCID-CIEF process,and verified for the specificity,accuracy,repeatability,intermediate precision,durability and inter-batch consistency.Results The optimal focusing time of WCID-CIEF for the determination of PRN antigen pI was 1 min at 1 500 V and 3 min at 3 000 V. The optimal final concentration of PRN antigen was 300 μg/mL. The PRN antigen pI was about 6. 035,and the blank matrix showed no interference peak in the position of each peak of antigen. The method had good specificity,accuracy,repeatability,intermediate precision,durability and consistency among batches.Conclusion The developed WCID-CIEF method is suitable for the pI detection and charge heterogeneity analysis of PRN antigen,which can provide basis for the characterization of PRN antigen and reference for the quality control in the process of development and production of related vaccines.
RESUMO
@#Objective To establish and verify a capillary isoelectric focusing-whole column imaging detection(CIEFWCID) method for the analysis of isoelectric point(pI) of recombinant human brain natriuretic peptide.Methods The ampholyte,space-occupying agent,protein concentration,focusing time were optimized by CIEF-WCID method,and the best condition for the detection of recombinant human brain natriuretic peptide was obtained.The repeatability,precision and durability of the developed method were verified,and three batches of recombinant human brain natriuretic peptide produced continuously were analyzed for pI.Results HR AESlyte 8-10.5 was selected as ampholyte,while 25 mmol/L sodium hydroxide as the space-occupying agent;The final concentration of the sample was 87.5 μg/mL and the focusing time was 8min.The relative standard deviation RSD of pI detection was 0.1% after six consecutive injections of the same sample;The RSD of pI detection of six samples was 0.1%;The pI RSD of the main peak was 0.1% at different final concentrations of the sample,and the pI RSD of the sample was 0.1% at different storage time,while the pI markers could not be changed arbitrarily.The pI was detected in three consecutive batches of recombinant human brain natriuretic peptide samples.Conclusion The developed CIEF-WCID method for pI analysis of recombinant human brain natriuretic peptide had good repeatability and precision and might be used for follow-up quality control of recombinant human brain natriuretic peptide.