Comparison of different ligand densities in immunoaffinity chromatography of the plantibody HB-01 coupled to Sepharose CL-4B to purify the rHBsAg.

This paper evaluates the immunopurification behavior of a plantibody HBsAg specific plantibody coupled to Sepharose CL-4B at different ligand densities. Results show no significant differences in the adsorption and elution capacities, and rHBsAg recovery of immunosorbents at 3.43, 4.45, and 5.31 mg/mL of ligand densities compared to its mouse-derived mAb counterpart consistently used in the rHBsAg purification process. Therefore, plantibody ligand densities higher than 3.43 mg/mL do not improve the immunopurification behavior of this immunosorbent, but increase the antibody consumption and the Hepatitis B vaccine cost. Immunosorbent of 2.23 mg/mL of ligand density demonstrated a poor performance. The IgG leached detectable level never exceeded the approved limit (3 ng IgG/microg rHBsAg). Values close to this limit were only observed at the ligand density of 5.31 and 2.27 mg/mL. In the case of the ligand density of 2.23 mg/mL the IgG leached value was high (2.90 ng IgG/microg rHBsAg) due to a low level of eluted antigen. In conclusion, it supports feasibility of using this plantibody at 3.43 mg/mL of ligand density for large-scale immunopurification of rHBsAg for human use, avoiding the biosafety and ethical concerns of the massive use of animals for this purpose.

Hepatitis B surface antigen immunopurification using a plant-derived specific antibody produced in large scale.

This paper provides an evaluation of a plant-derived HBsAg-specific antibody in the immunopurification of the recombinant HBsAg for vaccine purposes. This plant-derived antibody was obtained from different batches of 100-200kg of tobacco leaves and coupled to Sepharose CL-4B with high efficiency. The plant-derived antibody immunoaffinity matrix purification behavior (elution capacity, antigen purity, purification cycles, and ligand leakage) was comparable to that of its mouse-derived monoclonal antibody homolog. This result supports the feasibility of using this plant-derived antibody for the immunopurification of the Hepatitis B surface antigen for human use, opening a new possibility to overcome the constrain of monoclonal antibody production in mice.

Large-scale purification of an antibody directed against hepatitis B surface antigen from transgenic tobacco plants.

The application of bioengineering to plants for production of biological products for human and animal use has expanded in recent years. The reasons for this expansion are several and include advances in the technology for novel production systems and the need for very large quantities of therapeutic proteins. The process of growing pharmaceutical proteins in plants, extracting, and purifying is a hard task considering the lack of available information concerning these topics. In this work, a recombinant murine monoclonal antibody specific for the hepatitis B surface antigen, expressed in stably transformed transgenic Nicotiana tabacum plants, was purified by means of a recombinant protein A Streamline chromatography as the main purification step. The antibody expression level varied with the age of the plants and the number of harvests from 40 to 15microg/ml and the maximum process yield was about 25mg of plantibody/kg of biomass. Protein A Streamline chromatography was successfully used in the purification process yielding a recovery of about 60% and a plantibody SDS-PAGE purity of over 90% but unexpectedly, previous clarification steps could not be totally avoided. The amino acid sequence recognized by this affinity purified plantibody was similar to its murine counterpart verifying the potentiality of plants to replace animals or bioreactors for large-scale production of this monoclonal antibody.

A very sensitive enzyme-linked immunoabsorbent assay to Staphylococcal protein A in the presence of immunoglobulins.

The production of recombinant hepatitis B virus surface antigen (rHBsAg) purified by immunoaffinity chromatography with monoclonal antibodies is used to obtain a vaccine against this virus. Monoclonal antibodies to rHBsAg from mouse ascites have been purified by Staphylococcal Protein A (SpA)--prior coupling to Sepharose CL-4B (Amersham-Bioscences, Uppsala, Sweden). A high sensitivity immunoassay has been developed for the quantification of part-per-million of SpA contaminants likely to co-purify with monoclonal antibodies obtained by Protein A affinity chromatography, in the presence of immunoglobulins. Specific sheep polyclonal Abs against SpA (SpAc1) were used as plate coating and the SpA detection was possible thanks to the conjugates of sheep Ab fragments F(ab)(2) (fSpAc1) and horseradish peroxidase (fSpAc1-peroxidase), reducing the possible unspecific interaction between SpA and Fc fragments. The immunoassay was shown to be specific for SpA contaminants. The quantification limit of the assay was 0.39 ng/ml spreading to the measurement of contamination levels less than 2 ppm of SpA in final preparations of monoclonal antibodies used for the immunopurification of pharmaceutical products, which is quite low for this application.

Procurement of reference materials against recombinant HBsAg surface antigen.

Mabs against HBsAg have been used for structural analyses, development of diagnostic tests, and for antigen immunopurification. Resultant products obtained from current methods of genetic recombination demand reference materials to test their potency, identity, purity, and the biological and immunological specific activity corresponding to their manufacturing processes. In this paper, we present a method for the qualitative and quantitative characterisation of CB.Hep-1 and CB.Hep-4 RM and their stability, in real time, with the required quality to be used as primary reference materials. Among the criteria applied in this study, we considered Mab-specific concentration through ELISA, purity, and total proteins by various methods, quantification of antigen recognition capacity of Mabs through the Ag-Mab absolute-recognition method. Sterility, homogeneity, stability, subclass, and isoelectric focusing were used in the characterisation of the reference materials.