Quantitative detection of residual porcine host cell DNA by real-time PCR.

All biological products are derived from complex living systems and are often mixed with large numbers of impurities. For reasons of safety, residual host-cell DNA must be eliminated during processing. To assay host-cell DNA content in biopharmaceutical products derived from porcine sources, this study applies the quantitative real-time polymerase chain reaction (Q-PCR) method. The optimized assay in this study is based on the pol region of the porcine endogenous retrovirus (PERV). Assay validation results demonstrate that the proposed assay has appropriate accuracy, preciseness, reproducibility, and sensitivity. Primer and probe specificity are evaluated in real-time Q-PCR reactions using genomic DNA from rabbit, mouse, cat, hamster, monkey, human cell, yeast, and Escherichia coli as templates. The sensitivity of real-time Q-PCR is determined using genomic DNA from the porcine kidney cell line. The reliable detection range is within 0.5-10(5) pg/reaction. The limit of quantitation is 500 fg. The sensitivity of the assay meets the authority criterion. Moreover, the assay is applied to determine the level of host-cell DNA in recombinant human coagulation factor IX (rhFIX) from transgenic pigs. The real-time Q-PCR assay is thus a promising new tool for quantitative detection and clearance validation of residual porcine DNA when manufacturing recombinant therapeutics.

Inactivation strategy for pseudorabies virus in milk for production of biopharmaceuticals.

UNLABELLED: By selecting pseudorabies virus (PrV) as a model virus, this study assessed the feasibility of applying viral inactivation strategies to manufacturing medicinal products from the milk of transgenic sows. The efficacy of heat, acidic/alkaline and detergent treatments was also evaluated with respect to their ability to inactivate PrV in milk samples. Experimental results indicate that PrV was inactivated obviously at least 7.125 log10 for 30 min at 60 degrees C. At alkaline values of pH 10 and acidic value of pH 4, PrV infectivity was reduced to 3.625 log10 and exceeded 5 log10, respectively. Moreover, PrV virus was inactivated efficiently (> 3.875 log10) by using 0.25-1% of Triton X-100 treatment and without a loss of biological activity of the recombinant human coagulation factor IX (rhFIX). RESULTS: of this study demonstrate the effectiveness of the proposed detergent inactivation method for PrV inactivation of rhFIX production from transgenic products, especially in milk materials.

Characterization of a monoclonal antibody specific to the Gag protein of porcine endogenous retrovirus and its application in detecting the virus infection.

The porcine endogenous retrovirus (PERV) has drawn extensive attention recently, due to the widespread use of biomaterials of porcine origin in organ transplantation. This virus is present in all pig strains and has been demonstrated to be capable of infecting human cells in vitro. Therefore, it is imperative to develop a highly sensitive and specific immunoassay for clinical surveillance in patients receiving xenotransplantation. We describe here the generation of a monoclonal antibody (mAb) named A-11 specifically against the Gag protein of PERV. The mAb was found to be able to detect PERV produced from cultured cells. No cross-reaction with Gag proteins of murine leukemia virus (MuLV) and human immunodeficiency virus-1/2 was observed indicating that it is highly specific to PERV. The mAb was characterized as IgG2b subtype and kappa light chain. The region recognized by the mAb A-11 was localized to amino acid 293-336 on the Gag protein, and a synthetic peptide corresponding to amino acid 313-322 effectively competed the binding of the mAb with recombinant Gag proteins. Both immunocytochemistry and flow cytometry showed that the antibody is suitable for detection of PERV infection. By using the assays, we found that PERV-infected cells primarily of epithelial origin, with the highest infection rate in 293 followed by HEp-2 cells. In summary, the A-11 mAb will be useful for the development of quantitative and qualitative immunoassays for monitoring PERV infection in xenotransplantation patients and individuals who have close contact with pigs.

Establishing the reactivity of monoclonal antibodies against porcine endogenous retrovirus envelope protein.

Xenotransplantation of pig organs may be associated with a risk of transmission of microorganisms. Porcine endogenous retroviruses (PERV) are of particular concern since in vitro experiments have demonstrated that human cells are susceptible to such microorganisms. To monitor the transmission of PERV, highly sensitive and specific immunoassays must be developed for clinical surveillance. This report describes the production, preliminary characterization and application of a monoclonal antibody (mAb) against a recombinant PERV envelope (Env) protein. The generated mAb was tested using recombinant PERV Env protein expressed in Escherichia coli, purified PERV virus particles and human 293 cell line infected with PERV. PERV-translated proteins of 15, 70 and 85 kD were recognized specifically using PERV-8E10 mAb and Western blotting. No cross-reactivity was demonstrated with exogenous viral protein (HIV, HTLV and MuLV). Moreover, PERV-8E10 mAb can be applied to localize PERV proteins using an immunoperoxidase assay. This work reveals that recombinant PERV Env protein and mAb may be effective in detecting antibodies against PERV in xenotransplanted patients, or for butchers who have extensive contact with pigs.