Background: Although successful, the second-generation hepatitis B vaccine programs around the world have a small group of immunized individuals that does not respond efficiently to the vaccination. Other issues of these vaccines are individuals that are low or nonresponders and/or have incomplete protection against heterologous hepatitis B virus (HBV) genotypes/subtypes and against HBV escape mutants. In addition, there are approximately 240 million people chronically infected with HBV worldwide and 620,000 deaths per year caused by the infection. Methods: In this study we developed three Hansenula polymorpha plasmids containing the following sequences: (a) HBsAg subtype ayw, (b) HBcAg sequence subtype adw2, and (c) chimeric HBsAg (adw4/ ayw) - preS1 (adw2) - 3 repetitions of preS2 (genotypes A, B, and C). The sequences were successfully expressed and the antigens purified. Using Balb/c mice the antigens were tested in different dosage combinations. Results: Three antigens were obtained at a high purity level and with high reproducibility. We also assessed their immunogenic properties, showing that the antigens, individually or in combination, generated anti-HBs, anti-preS1, anti-preS2, and anti-HBc antibodies efficiently in mice. Conclusions: The formulation tests showed that a combination of 0.02 μg of HBs, 0.2 μg of preS1-preS2-HBs, and 0.02 μg of HBc was effective in eliciting specific antibodies in mice.

In this work, several chromatographic methods were studied for the purification of recombinant clotting factors VII (FVIIr) and VIII (FVIIIr) derived from human SK-Hep cell lines. The FVIIIr is used for the treatment of Hemophilia A, while the FVIIr is used for the treatment of Hemophilia B and Hemophilia A. Producing these factors in human cell lines results in glycosylation, sulphation and folding patterns similar to the endogenous factors produced in the human organism. Purification of FVIIIr by multimodal chromatography techniques using CaptoMMC resin, affinity using FVIIISelect resin and ion exchange (SP-Sepharose) yielded a fairly homogeneous and well-defined band profile (by SDS-PAGE) which demonstrated the expected presence of the light and heavy chains, Westen-Blott indicated that commercial antibodies recognized the heavy chain of the studied molecule. The techniques allowed a high reproducibility of the process where purification sequences indicated the same behavior of chromatographic profiles and the process eliminated 99.5% ± 0.5% nonspecific proteins and recovering up to 64% FVIIIr. FVIIr was purified with only a single chromatographic technique using the FVIISelect resin which isolated the protein by removing about 99% impurities and recovering virtually the entire product. The affinity chromatography eluate was dialyzed on 5 kDa membranes which resulted in the autoactivation process of the FVIIr molecule resulting in a signal increase of up to 5 fold over the initial amount. The SDS-PAGE gel and Westen-Blott demonstrated the auto-activation process where a migration of 50 kDa to 30 kDa band was observed and the commercial antibodies against FVII were able to detect the band. The purification method was also quite reproducible and the band profile very similar compared to the commercial products. Thus, it was possible to obtain purification platforms for the FVIIr and FVIIIr proteins.

Coagulation factor VIII is one of the largest proteins attempted to be expressed in recombinant form. A very complex and labile protein which has a very short half-live and need a fast and efficient purification chain. Here, we describe a simple purification sequence using multimodal Capto MMC, affinity FVIII select and ion exchange SP-Fastflow chromatography steps without subjecting the target molecule to mechanical and temperature stress, separating impurities from rFVIII using net charge, hydrophobicity, and affinity of the molecules.

Recombinant coagulation factor VII is a very important and complex protein employed for treatment of hemophiliac patients (hemophilia A/B) who develop inhibitors antibodies to conventional treatments (FVIII and FIX). The rFVII is a glycosylated molecule and circulates in plasma as zymogen of 50 kDa. When activated the molecule is cleaved to 20-30 kDa and has a half-life of about 3 h, needing to be processed fast and efficiently until freeze-drying. Here, we describe a very simple and fast purification sequence for rFVII using affinity FVII Select resin and a dialysis system that can be easily scaled up.