12
views
0
recommends
+1 Recommend
1 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: found
      Is Open Access

      Validación de un ELISA para la cuantificación de las impurezas proteicas de la cepa hospedera en el principio activo de la vacuna recombinante cubana contra la hepatitis B Translated title: Validation of an ELISA for the quantification of protein impurities from host strain on the active principle of the Cuban recombinant vaccine against hepatitis B

      Read this article at

      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          Se validó un inmunoensayo tipo sandwich de doble anticuerpo para cuantificar las impurezas proteicas de la cepa hospedera que pueden estar presentes en el principio activo de la vacuna cubana contra la hepatitis B. Se prepararon los reactivos biológicos empleados en el ELISA. Las proteínas de la cepa hospedera se obtuvieron bajo las mismas condiciones que el proceso de producción de la proteína recombinante hasta un paso de semipurificación o purificación primaria. Los antisueros dirigidos contra las proteínas de la cepa hospedera se generaron en conejos por un proceso de inmunización en cascada. Para la validación se analizaron los siguientes parámetros: linealidad, límite de detección y cuantificación, exactitud, rango y precisión. El ensayo establecido resultó específico, con una exactitud entre 89% y 109% para todos los tampones estudiados; se demostró un ajuste parabólico y un rango de trabajo entre 0,63 y 1,25 ng/mL, la repetibilidad y la precisión intermedia mostraron coeficientes de variación inferiores al 10 y 20% respectivamente.

          Translated abstract

          A double sandwich immunoassay was validated to quantify antibody protein impurities from host strain that can be present in the active pharmaceutical ingredient of Cuban vaccine against hepatitis B. All biological reagents used in the ELISA were prepared and characterized. The proteins from the host strain were obtained under the same conditions as the production process of the recombinant protein until a purification or primary semipurification step. The antisera addressed against those proteins were generated in rabbits by an immunization process in cascade. For validation the parameters analyzed were: linearity, limit of detection and quantification, accuracy, range and precision. The established assay was specific, and the calculated accuracy was from 89% to 109% for all studied buffers. A parabolic fit and a working range from 0.63 to 1.25 ng/mL were demonstrated. The repeatability and intermediate precision showed variation coefficients below 10% and 20% respectively.

          Related collections

          Most cited references 21

          • Record: found
          • Abstract: not found
          • Conference Proceedings: not found

          Validation of a analytical procedures : text and methodology Q2(R1)

          (2005)
            Bookmark
            • Record: found
            • Abstract: not found
            • Article: not found

            Validation of Analytical Procedures: Text and Methodologies Q2(R1)

              Bookmark
              • Record: found
              • Abstract: found
              • Article: not found

              Analysis for residual host cell proteins and DNA in process streams of a recombinant protein product expressed in Escherichia coli cells.

              Analyses of crude samples from biotechnology processes are often required in order to demonstrate that residual host cell impurities are reduced or eliminated during purification. In later stages of development, as the processes are further developed and finalized, there is a tremendous volume of testing required to confirm the absence of residual host cell proteins (HCP) and DNA. Analytical tests for these components are very challenging since (1). they may be present at levels that span a million-fold range, requiring substantial dilutions; (2). are not a single component, often existing as fragments and a variety of structures; (3). require high sensitivity for final steps in process; and (4). are present in very complex matrices including other impurities, the product, buffers, salts and solvents. Due to the complex matrices and the variety of potential analytes, the methods of analysis are not truly quantitative for all species. Although these limitations are well known, the assays are still very much in demand since they are required for approval of new products. Methods for final products, described elsewhere, focus on approaches to achieve regulatory requirements. The study described herein will describe the technical rationale for measuring the clearance of HCP and DNA in the entire bioprocessing to purification from an Escherichia coli-derived expression system. Three analytical assays, namely, reversed-phase high-performance liquid chromatography (RP-HPLC), enzyme-linked immunosorbent assay (ELISA), and Threshold Total DNA Assay, were utilized to quantify the protein product, HCP and DNA, respectively. Product quantification is often required for yield estimation and is useful since DNA and HCP results are best expressed as a ratio to product for calculation of relative purification factors. The recombinant E. coli were grown to express the protein of interest as insoluble inclusion bodies (IB) within the cells. The IB were isolated by repeated homogenization and centrifugation and the inclusion body slurry (IBS) was solubilized with urea. After refolding the product, the solution was loaded on several commonly used ion exchangers (CM, SP, DEAE, and Q). Product was eluted in a salt gradient mode and fractions were collected and analyzed for product, HCP and DNA. The IBS used for this study contained about 15 mg/ml product, 38 mg/ml HCP and 1.1 mg/ml DNA. Thus, the relative amounts of HCP and DNA in the IBS was excessive, and about 10(3) times greater than typical (because the cells and IB were not processed with the normal number of washing steps during isolation). This was of interest since similar samples may be encountered when working with non-inclusion body systems, such as periplasmic expressions, or in cases where the upstream unit operations under-perform in IB cleaning. The study described herein describes the development of three robust methods that provide the essential process data needed. These findings are of general interest to other projects since applications of similar analytical technology may be used as a tool to develop processes, evaluate clearance of impurities, and produce a suitable product.
                Bookmark

                Author and article information

                Contributors
                Role: ND
                Role: ND
                Role: ND
                Role: ND
                Role: ND
                Role: ND
                Role: ND
                Journal
                vac
                Vaccimonitor
                Vaccimonitor
                Finlay Ediciones (Ciudad de la Habana )
                1025-0298
                April 2014
                : 23
                : 1
                : 11-16
                Affiliations
                [1 ] Centro de Ingeniería Genética y Biotecnología Cuba
                Article
                S1025-028X2014000100003
                Product
                Product Information: website
                Categories
                BIOLOGY

                General life sciences

                validación, hepatitis B, vaccine, validation, ELISA, vacuna

                Comments

                Comment on this article