Miyares M, González J, Torres D, Muñoz L, Pestana Y, Padrón S, Herrera I, Rosales I, Linares M

Language: Spanish
References: 9
Page: 14-18
PDF size: 223.96 Kb.

ABSTRACT

A formulation was obtained for a vaccinal candidate of hepatitis C virus (VHC) in the CIGB. It is a plasmid for the immunization with DNA contianing the genes of the three structural antigens of the virus, blended with a recombinant truncated variant of the protein of the core of the VHC, as molecular adjuvant. Both compounds were formulated in separate vials and were mixed at the moment of the immunization. In the laboratory of Development-analytic the technique of determination of total proteins was validated by the microcoomassie method for the sample of Finished Product (FP) of Hepatitis C core antigen (HCVcoreAg). The validated parameters were: Specificity, Linearity and Range, Accuracy, Precision, Stability of the FP sample under two different conditions of storage. The method was specific for the quantification of the HCcAg without interferences of sample placebo of FP to superior dilutions at 1:8. The curve of the microcoomassie for the FP was lineal in the range of work of 5-40 µg/mL. It demonstrated the accuracy of the method (100±10% recovery). In the evaluation of the system, the established acceptance criteria were completed for Precision: Repeatability (CV ≤ 5%) and Intermediate Precision (CV ≤ 10%). Temperatures studied in stability did not cause alteration in HCcAg concentration, and the FP can be stored for 15 days. Validation was satisfactory. The method is adequate to quantify the sample of the FP. This result allows us to comply with the specifications established for this product.

REFERENCES

1. Francki RIB, Fauquet CM, Knudsen DL, Brown F, editores. Classification and nomenclature of viruses. Fifth report of the International Committee on Taxonomy of Viruses. Archives of Virology, suppl. 2. New York: Springer-Verlag; 1991.

2. Choo QL, Richman KH, Han JH, Berger K, Lee C, Dong C, et al. Genetic organization and diversity of thehepatitis C virus. Proc Natl Acad Sci 1991;88:2451-5.

3. Álvarez-Lajonchere L, Guerra I, Musacchio A, Aguilar JC, Falcon V, Soria Y, et al. Recombinant in vitro assembled hepatitis C virus core particles induce strong specific immunity enhanced by formulation with an oil-based adjuvant. Biol Res 2009;42(1):41-56.

4. Grakoui A, Wychowski C, Lin C, Feinstone S, Rice CM. Expression and identification of hepatitis C virus polyprotein cleavage products. J Virol 1993;67:1385-95.

5. Tsaffrir Zor, Zvi Selinger. Linearization of the Bradford Protein Assay Increasses Its Sensitivity: Theorical and Experimental Studies. Analytical Biochemistry 1996;236:302-08.

6. Chaloner-Larsson G, Anderson R, Egan A. A WHO guide to good manufacturing practice (GMP) requirements. Part 2: Validation. Validation of analytical assays. Geneva: WHO;1997.

7. Steven Walfish. Analytical Methods: A Statistical Perspective on the ICH Q2A and Q2B Guidelines for Validation of Analytical Methods. BioPharm International 2006;19(12). Disponible en: http://www.biopharminternational.com/biopharm/article/ articleDetail. jsp?id=392483

8. Centro para el Control Estatal de la Calidad de los Medicamentos (CECMED). Validación de Métodos Analíticos. Regulación No.41. La Habana: CECMED; 2007.

9. Harmonised Tripartite Guideline (ICH). Validation of analytical procedures: Text and Methodology. London: ICH; 2005.