Viral inactivation plays a critical role in assuring the safety of monoclonal antibody (mAb) therapeutics. Traditional viral inactivation involves large holding tanks in which product is maintained at a target low pH for a defined hold time, typically 30-60 min. The drive toward continuous processing and improved facility utilization has provided motivation for development of a continuous viral inactivation process. To this end, a lab-scale prototype viral inactivation system was designed, built, and characterized. Multiple incubation chamber designs are evaluated to identify the optimal design that enables narrow residence time distributions in continuous flow systems. Extensive analysis is conducted supporting rapid low pH viral inactivation and included evaluations with multiple viruses, a range of pH levels, buffer compositions, mAb concentrations, and temperatures. Multiple test conditions are evaluated using the in-line system and results compared to traditional batch-mode viral inactivation. Comparability in kinetics of virus inactivation suggests equivalency between the two approaches.

Purpose: The poor immunogenicity of tumor antigens and the antigenic heterogeneity of tumors call for vaccine strategies to enhance T-cell responses to multiple antigens. Two antigens expressed noncoordinately on most human carcinomas are carcinoembryonic antigen (CEA) and MUC-1. We report here the construction and characterization of two viral vector vaccines to address these issues. Experimental Design: The two viral vectors analyzed are the replication-competent recombinant vaccinia virus (rV-) and the avipox vector, fowlpox (rF-), which is replication incompetent in mammalian cells. Each vector encodes the transgenes for three human costimulatory molecules (B7-1, ICAM-1, and LFA-3, designated TRICOM) and the CEA and MUC-1 transgenes (which also contain agonist epitopes). The vectors are designated rV-CEA/MUC/TRICOM and rF-CEA/MUC/TRICOM. Results: Each of the vectors is shown to be capable of faithfully expressing all five transgenes in human dendritic cells (DC). DCs infected with either vector are shown to activate both CEA- and MUC-1–specific T-cell lines to the same level as DCs infected with CEA-TRICOM or MUC-1-TRICOM vectors. Thus, no evidence of antigenic competition between CEA and MUC-1 was observed. Human DCs infected with rV-CEA/MUC/TRICOM or rF-CEA/MUC/TRICOM are also shown to be capable of generating both MUC-1- and CEA-specific T-cell lines; these T-cell lines are in turn shown to be capable of lysing targets pulsed with MUC-1 or CEA peptides as well as human tumor cells endogenously expressing MUC-1 and/or CEA. Conclusion: These studies provide the rationale for the clinical evaluation of these multigene vectors in patients with a range of carcinomas expressing MUC-1 and/or CEA.

Two closely-linked genes, Ragl and Rag2, are required for the recombination of V, D, and J gene segments to form the genes encoding V regions of Igs and T-cell receptors. In several mammalian species, chickens, and Xenopus laevis, the coding region of RAG1 lies within a single exon; its sequence is highly conserved from amphibians to mammals (Schatz et al. 1992;Oettinger 1992). These features suggest that it may be possible to use the polymerase chain reaction (PCR) with degenerate primers to amplify a segment of Rag1 from genomic DNA of more primitive species.When zebrafish (Danio rerio) DNA was PCR-amplified under conditions similar to those used previously forXenopus Ragl (Greenhalgh et al. 1993), with the same degenerate primers, a product of expected size [638 base pairs (bp)] was obtained. Although amplification of nurse shark (Ginglymostoma cirratum) DNA yielded no product of this size detectable by ethidium bromide staining, a faint band migrating at the expected position was detected by Southern blotting with a Xenopus Rag1 probe. This product was gel-purified and reamplified with the same reverse primer and a forward primer internal to that used initially: 5'-AARATGAARCCNGTNTGG-3'. A product of expected size (359 bp) was obtained. The zebrafish and shark amplification products were cloned and sequenced on both strands. Southern blotting of Eco RI digests of genomic DNA, at high stringency, with probes derived from the cloned zebrafish and shark products revealed bands of N0.9 and 1.0 kb, respectively. Comparison of the nucleotide and predicted amino acid sequences of the zebrafish and shark RAG1 segments with sequences of the corresponding segments of mouse, chicken, and Xenopus Ragl shows a high degree of conservation (Fig. 1). The amino acid sequence encoded by the zebrafish segment is 74%-79% identical to that in Xenopus, chicken, or mouse; in this region the identity among the latter three sequences is 90%. The amino acid sequences corresponding to the shark segment are even more conserved. Particularly striking is the conservation from positions 143 to 186, in which the amino acid sequence of Ragl in all five species is identical except for a single Ile ~ Val replacement in the zebrafish sequence.Relatively little is known about lymphopoeisis in teleosts or elasmobranchs. The availability of specific nucleic acid probes for Ragl in zebrafish and shark should facilitate the identification of sites of V (D) J rearrangement in B and T lymphocytes. Rag1 may also be an indicator of the presence of rearranging immune system genes in cyclostomes, primitive vertebrates in which immunoglobulins and T-cell receptors have not yet been clearly identified. However, our attempts to identify Ragl in lampreys and hagfish by PCR amplification and by Southern blotting have not been successful.

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BACKGROUND Virus inactivation of plasma products is conducted using stainless‐steel vessels. Single‐use technology can offer significant benefits over stainless such as operational flexibility, reduced capital infrastructure costs, and increased efficiency by minimizing the time and validation requirements associated with hardware cleaning. This study qualifies a single‐use bag system for solvent/detergent (S/D) virus inactivation. STUDY DESIGN AND METHODS Human plasma and immunoglobulin test materials were S/D‐treated in Mobius single‐use bags using 1% tri‐n‐butyl phosphate (TnBP) with 1% Triton X‐100 or 1% Tween 80 at 31°C for 4 to 6 hours to evaluate the impact on protein quality. Volatile and nonvolatile organic leachables from low‐density polyethylene film (Pureflex film) used in 1‐L‐scale studies after exposure to S/D in phosphate‐buffered saline were identified compared to controls in glass containers. Virus inactivation studies were performed with xenotropic murine leukemia virus (XMuLV) and bovine viral diarrhea virus (BVDV) to determine the kinetics of virus inactivation, measured using infectivity assays. RESULTS S/D treatment in Mobius bags did not impact the protein content and profile of plasma and immunoglobulin, including proteolytic enzymes and thrombin generation. Cumulative leachable levels after exposure to S/D were 1.5 and 1.85 ppm when using 0.3% TnBP combined with 1% Tween 80 or 1% Triton X‐100, respectively. Efficient inactivation of both XMuLV and BVDV was observed, with differences in the rate of inactivation dependent on both virus and S/D mixture. CONCLUSION Effective S/D virus inactivation in single‐use container technology is achievable. It does not alter plasma proteins and induces minimal release of leachables.