ABSTRACT
The study was designed to establish a 2D UPLC-QTOF method to extrapolate the structure of an unknown substance in carboplatin injection and its relationship with the excipient. By using phenyl-hexyl column (250 mm×4.6 mm, 5 μm) with mobile phase consisting of tetrabutylammonium sulfate buffer (pH 7.5) and acetonitrile in gradient elution mode, an unknown impurity in carboplatin injection was found and quantitatively determined. Then a 2D UPLC-QTOF, HSS T3 column (100 mm×2.1 mm, 1.7 μm) was employed to confirm the molecular weight and the structure of the unknown impurity (electrospray ionization source, positive ion mode, MSE mode) with mobile phase consisting of 0.1% formic acid and acetonitrile. The relationship among impurities, API and excipient was investigated by accelerated stability test with ICP-MS/MS, ICP-AES. Results showed that disodium edetate in the formulation interacted with carboplatin producing an unknown impurity containing platin, and induced the increase of 1,1-cyclobutanedicarboxylic acid. The research should be done on the rationality of the addition of disodium edetate in such injections containing heavy metals.
ABSTRACT
The M protein gene of porcine reproductive and respiratory syndrome virus amplified by PCR was tandem linked with its GP5 gene in shuttle vector in correct frame, resulting in shuttle vector pShuttle-CMV-M-GP5. The positive clone was identified by PCR and further confirmed by sequencing. The constructed plasmid was linearized with Pme I and co-transformed BJ5183 host bacteria with pAdEasy-1 to produce recombinant adenovirus DNA by homologous recombination. Then the adenovirus DNA was linearized with Pac I and transfected into HEK-293A cells to obtain recombinant adenovirus. The specific expression of target proteins by the recombinant adenovirus was verified by indirect immuno-fluorescence assay (IFA) with monoclonal antibodies against M and GP5.The results showed that the tandem linked M with GP5 could be co-expressed by adenovirus vector. Mice immunized with the constructed recombinant adenovirus induced strong humoral immunity (ELISA antibody and virus neutralizing antibody) and cellular immunity (lymphocyte proliferation and CTL responses). The results showed that the recombinant adenovirus has strong immunogenicity and provided the basis for the further experiments in pigs.