ObjectivesTo develop and optimize quantitative HPLC method using 2,3-naphthalenedicarboxaldehyde (NDA) after simple and efficient solid phase extraction to determine the histamine in a biopharmaceutical (Histobulin™).MethodsThe HPLC method was established using NDA-induced Histobulin and compared with the recently reported HPLC method using o-phthaldehyde (OPA). The validated NDA-applied HPLC method was adjusted to 15 lots of Histobulin and compared by the current lot-release-test method using fluorimetry in recovery of histamine and reproducibility.ResultsAnalyses of six HPLC chromatograms using NDA and OPA each were compared. NDA produced a more stable chromatogram baseline than OPA, and showed better stability. The HPLC analysis was validated in accuracy (91–103%), precision (interday/intraday assay CV ≤2.30%), and linearity of dose–response curve (R2 ≥ 0.9919). The detection limit was 0.0076 μg/mL and the quantitative limit was 0.0229 μg/mL. The amount of histamine per 12 mg of immunoglobulin was determined to be 0.17 ± 0.016 μg by the HPLC and 0.025 ± 0.013 μg by the current lot-release-test method using fluorimetry.ConclusionNDA derivatization showed better stability compared with the OPA method. Therefore the newly established NDA-derivatizated HPLC method may be more suitable than the fluorimetric method in lot-release-tests of biopharmaceuticals.

The attenuation and immunoenhancing effects of rpoS and phoP Salmonella enterica serovar strain Typhi (Salmonella typhi) mutants have not been compared. Here, three S. typhi deletion mutants (phoP, rpoS, and rpoS-phoP double mutant) are constructed and these mutants are characterized with respect to invasiveness, virulence, and protective immune response compared with wild-type Ty2. It was found that phoP and phoP-rpoS deletion mutants are less invasive to HT-29 cells than the wild-type Ty2 and the rpoS single-deleted strain. The LD(50) of immunized mice was higher for phoP than for rpoS mutants, and the highest for the phoP-rpoS double mutant. In addition, all S. typhi mutants showed an increase in the specific serum IgG levels and T-cell-mediated immunity, and showed equal protection abilities against a wild-type Ty2 challenge after two rounds of immunization in BALB/c mice. It is concluded that phoP genes appear to play a more important role than rpoS genes in both cellular invasion and virulence of S. typhi, but not in immunogenicity in mice. Furthermore, the data indicate that the phoP-rpoS double mutant may show promise as a candidate for an attenuated typhoid vaccine.

Recently, the genetic modification of mesenchymal stem cells (MSCs) has led to increased differentiation potential. For the therapeutic application of genetically modified MSCs, it is crucial to evaluate their characteristics and safety. In this study, we investigated the effects of bone morphogenetic protein 2 (BMP2) gene transfer on the characteristics and biodistribution of human MSCs. Lentiviral-mediated BMP2 transduction to MSCs enhanced osteocyte differentiation and decreased adipocyte differentiation. Although there is no significant difference in cell proliferation capacity, MSCs transduced BMP2 proliferate somewhat higher than nontransduced or GFP transduced MSCs. No significant changes were observed in surface antigen expression in genetically modified MSCs. In vivo transplantation of lentiviral-mediated BMP2 gene transferred MSCs to nude mice did not result in tumor formation. To evaluate the biodistribution of genetically modified cells, MSCs carrying BMP2 were injected into the tail vein of femur fractured mice. The introduced MSCs were detected in the spleen, testis and fractured femur 28 days post-implantation. These findings suggest that diverse safety tests for genetically modified MSCs should be considered, particularly when a lentivirus mediated gene transfer method is used.

Influenza vaccine potency, which is determined by quantitatively measuring the content of Hemagglutinin (HA), is an essential index representing the efficacy of the vaccine. Standardization of the single radial immunodiffusion (SRID) assay, a method for measuring HA content, and proficiency of the testing institutions are crucial for influenza vaccine quality control. Herein, we assessed the proficiency of SRID assays at the National Control Laboratory (NCL) of Korea and several vaccine manufacturers. Eight laboratories participated in this study, and the proficiencies of all laboratories yielded satisfactory results in overall SRID assays. In contrast, there were some unsatisfactory results in measuring with different types of agarose gel plates produced by other laboratories. Overall, our findings demonstrated that the proficiency of SRID assay in the tested laboratories is acceptable for quality control of influenza vaccines and that detailed review on the validation reports regarding the test methods will be helpful for better control.