Application of a risk analysis method to different technologies for producing a monoclonal antibody employed in hepatitis B vaccine manufacturing.

CB.Hep-1 monoclonal antibody (mAb) is used for a recombinant Hepatitis B vaccine manufacturing, which is included in a worldwide vaccination program against Hepatitis B disease. The use of this mAb as immunoligand has been addressed into one of the most efficient steps of active pharmaceutical ingredient purification process. Regarding this, Quality Risk Management (QRM) provides an excellent framework for the risk management use in pharmaceutical manufacturing and quality decision-making applications. Consequently, this study sought applying a prospective risk analysis methodology Failure Mode Effects Analysis (FMEA) as QRM tool for analyzing different CB.Hep-1 mAb manufacturing technologies. As main conclusions FMEA was successfully used to assess risks associated with potential problems in CB.Hep-1 mAb manufacturing processes. The severity and occurrence of risks analysis evidenced that the percentage of very high severe risks ranged 31.0-38.7% of all risks and the huge majority of risks have a very low occurrence level (61.9-83.3%) in all assessed technologies. Finally, additive Risk Priority Number, was descending ordered as follow: transgenic plants (2636), ascites (2577), transgenic animals (2046) and hollow fiber bioreactors (1654), which also corroborated that in vitro technology, should be the technology of choice for CB.Hep-1 mAb manufacturing in terms of risks and mAb molecule quality.

A combinatorial strategy of a new monoclonal ELISA and immunoaffinity chromatography using sodium deoxycholate to increase the recovery of multimeric proteins like r-HBsAg.

In this work, a sandwich monoclonal-based ELISA for quantifying the HBsAg obtained from yeast cells was standardized and validated. The monoclonal antibody employed in this assay reacts uniformly with different molecular isoforms of r-HBsAg. Immunoassay allowed the r-HBsAg quantification in an analytical range 11.9-191.7 ng/mL. Inter- and intra-assay precision variation coefficients were between 0.77-3.43% and 1.95-8.89%, respectively, and the recovery ranged 98.2-100.8%; which confirms its reliability. r-HBsAg is a complex of carbohydrates, proteins and lipids assembled into spherical particles with an average diameter of 24 nm. Many host contaminants accompany this protein during purification process, which can interfere the antigen recognition by the immunoaffinity matrix. To solve this problem, the effect of several detergents in the quantification and purification of r-HBsAg were studied. The addition of the surfactant sodium deoxycholate (NaDoc) at 0.1% in this ELISA improved the recognition and quantification of r-HBsAg by 2.4-fold higher than untreated samples. Similar results were observed in the immunoaffinity chromatography where a 1.5-fold increasing recovery values was shown. The application of NaDoc allows to reduce the inhibitory effect upon the antigen-antibody recognition, increasing the quantification and immunoaffinity chromatography efficiency. This analytical combination could be applied to multimeric proteins like r-HBsAg of HB vaccine.

Prolonged stability of the plantibody HB-01 directed against the hepatitis B surface antigen in cryo-preserved tobacco leaves.

Since 1983, several recombinant antibodies have been expressed in important agronomic plant species. However, to date no evaluation has been published about prolonged antibody stability within plant tissues under cryo-preservation conditions. This current report presents an approach to the KDEL-plantibody HB-01 (PHB-01) stability in frozen tobacco leaves by presenting scientific evidence about the stability of a plantibody to a prolonged low temperature exposure in this biological source. Results clearly show that the PHB-01 amount is maintained during the storage of tobacco leaves at -20 degrees C for 90 days. The PHB-01 recovery was not affected by any irreversible physical and/or chemical change produced in tobacco leaves after this cryo-preservation time. The amount of total soluble proteins in the clarified extract decreased in proportion with the storage time and the PHB-01 molecules isolated from frozen leaf extracts were highly pure, >95%, according to an SDS-PAGE assessment under reducing conditions. Low temperature exposure of tobacco leaves did not reveal visible changes in frozen leaves, which is essential for the further extractability of proteins. The PHB-01 is stable in tobacco leaves at -20 degrees C during 90 days, which offers the possibility to overcome problems associated with detrimental climate conditions and optimize purification capabilities.

Purification process development for HER1 extracellular domain as a potential therapeutic vaccine.

HER1 is a tumor associated antigen emerging as an attractive target for cancer therapy. In the present study we demonstrated for first time that HER1 extracellular domain can be purified by a downstream process at pilot scale based on immunoaffinity chromatography from bioreactor supernatant of HEK 293 transfectomes. Filtered supernatant was applied to CNBr-activated Sepharose CL-4B with monoclonal antibody anti-human EGF immobilized, followed by three additional chromatographic polishing steps. HER1 extracellular domain was obtained with high purity (>95%), low DNA content, and biological activity.

Detection of Rubisco and mycotoxins as potential contaminants of a plantibody against the hepatitis B surface antigen purified from tobacco.

Antibodies have been one of the proteins widely expressed in tobacco plants for pharmaceutical purposes, which demand contaminant free preparations. Rubisco constitutes 40-60% of tobacco leaf soluble proteins; therefore it is the major potential protein contaminant of plantibodies, while mycotoxins are toxic compounds that could be introduced during the biomass production and post-harvest stages with important consequences to human health. The objective of this paper was to investigate whether Rubisco and mycotoxins are present in Plantibody HB-01 preparations used in the immunopurification of the hepatitis B surface antigen. Rubisco was purified from Nicotiana tabacum yielding 154 microg of protein per gram of leaves and purity over 95%. Among mouse monoclonal antibodies generated against this enzyme, the CBSS.Rub-2 was selected for its immunodetection. It recognizes a conserved sequential epitope of Rubisco large subunit with an affinity constant of 0.13 x 10(8)M(-1). Rubisco quantification limit was 1 microg spreading to the measurement of this contaminant less than 4% of plantibodies samples. Additionally, according to a Reverse Phase-HPLC used to measure the level of adventitiously introduced contaminants, it can be concluded that aflatoxins B1, B2, G1 and G2 were undetected in the purified Plantibody HB-01 samples.