Management of process technology.

A successful manufacturing scale cell culture process has as its basis a well-designed process. However, even the best designed large-scale process can become unreliable if not managed properly. The primary element to successful management of all large-scale processes is the careful attention to the details, especially the most important ones. The second element that requires serious consideration is the operation of that process within the confines of current regulatory environments. The major considerations are: 1. Adequate raw material specifications and testing. 2. Facility and equipment design that maximizes operational success and meets current regulatory requirements. 3. Thoroughly written procedures covering all aspects of operation and maintenance, and supported by a thorough documentation and review system. 4. A dedicated staff with a thorough understanding of systems and the importance of operational details. 5. Continual training and retraining programs. 6. Sound process and facility validation programs. 7. Comprehensive preventive maintenance programs. 8. Thorough understanding of process and facility trends, and the expected and acceptable ranges of results. 9. Development of sound procedures and practices that minimize batch losses and permit operation within current regulatory requirements. 10. Thorough and continual monitoring of all critical process parameters. 11. Process scheduling to minimize batch jeopardy or loss while maximizing throughout. 12. Maintenance of the appropriate environmental conditions of the plant and general cleanliness of plant and equipment. 13. Adequate batch loss prevention programs. 14. Adequate troubleshooting programs. 15. Adequate support organization and facilities both internally and externally to plant operations. With the appropriate application of sound process design and management principles, and with operational experience, large-scale cell culture process plants can be operated at success rates that exceed 95%.

Avian myeloblastosis provirus cloned in a lambda bacteriophage is leukemogenic.

The avian myeloblastosis virus provirus inserted in a lambda bacteriophage, recombinant clone 11A1-1 (Souza et al., Proc. Natl. Acad. Sci. U.S.A. 77:3004-3008, 1980), was transfected into chicken embryo fibroblasts which had been preinfected with either Rous-associated virus type 61 or the transformation-defective avian sarcoma virus tdB77. Within 4 to 5 h after transfection, the cells were injected into 16-day-old chicken embryos or 1-day-old chicks. Acute myeloblastic leukemia developed after a long latent period. Filtered (0.22-micrometer pores) supernatant of transformed buffy-coat cell cultures from one leukemic chicken of the lambda 11A1-1 (tdB77) group rapidly transformed yolk sac cells in vitro. Results from an infectivity interference assay and analysis of proviral DNA fragments generated with restriction endonucleases were consistent with the presence in leukemic cells of defective avian myeloblastosis virus and tdB77 as the helper virus.

Vertebrate DNAs contain nucleotide sequences related to the transforming gene of avian myeloblastosis virus.

Avian myeloblastosis virus contains a continuous sequence of approximately 1,000 nucleotides which may represent a gene (amv) responsible for acute myeloblastic leukemia in chickens. This sequence appears to have been acquired from chicken DNA and to be substituted for the envelope gene in the viral genome. We used hybridization probes enriched for the amv sequences and conditions that facilitate annealing of partially homologous nucleotide sequences to show that cellular sequences related to amv are present in the genomes of all vertebrates ranging from amphibians to humans but were not detected in fish, sea urchins, or Escherichia coli. In contrast to the preceding findings, nontransforming endogenous proviral nucleotide sequences closely related to the remainder of the avian myeloblastosis virus genome and to the entire myeloblastosis-associated helper virus are present only in chicken DNA. The amv-related cellular sequences appear to be highly conserved during evolution and to be contained at only one or a few locations in the genome of vertebrates. Within closely related species, they appear to share common evolutionary genetic loci. These findings and similar ones obtained with other highly oncogenic retroviruses containing a transforming gene suggest a general mechanism for acquisition of viral oncogenic sequences and an essential role for these sequences in the normal cellular state.

Production of R-type virus-like particles in hamster cells.

The production and release of R-type virus-like particles (VLP) was studied in several Syrian hamster cell lines. Most but not all hamster cell lines contain detectable levels of R-type VLP. However, only BHK-21 clone F (CF) cells would release the VLP into culture fluids. Treatment with dexamethasone enhanced to a limited extent the production and release of VLP from BHK-21 CF cells. Actinomycin D inhibited the production and release of R-type VLP in hamster cells, suggesting that some transcription from DNA is necessary for VLP production. Furthermore, 5-iododeoxyuridine induced the production of R-type VLP in Syrian hamster embryo cells. These results suggest that R-type VLP are endogenous to Syrian hamsters.

DNA of avian myeloblastosis-associated virus type 2 integrates at multiple sites in the chicken genome.

The cellular sites of integration of the avian myeloblastosis-associated virus type 2 (MAV-2) DNA have been examined by Southern blot analysis of cellular DNA from infected cloned and uncloned chicken embryonic fibroblasts. Provirus-cell juncture fragments were not detected in restriction enzyme digests of DNA from MAV-2-infected uncloned cells. However, each MAV-2-infected cell clone examined produced a unique set of junctive bands. Thse findings indicate that multiple sites of integration exists for MAV-2 proviruses in cellular DNA.

Characterization of avian myeloblastosis-associated virus DNA intermediates.

The major species of unintegrated linear viral DNA identified in chicken embryonic fibroblasts infected with either the avian myeloblastosis-associated viruses (MAV-1, MAV-2) or the standard avian myeloblastosis virus complex (AMV-S) has a mass of 5.3 X 10(6) daltons. An additional minor DNA component observed only in AMV-S-infected cells has a mass of 4.9 X 10(6) daltons. The unintegrated linear viral DNAs and integrated proviruses of MAV-1 and MAV-2 have been analyzed by digestion with the restriction endonucleases EcoRI and HindIII. MAV-2 lacks a HindIII site present in MAV-1. These fragments have been compared to those generated by EcoRI and HindIII digestion of linear viral DNAs of AMV-S. Restriction enzyme digestion of AMV-S viral DNA produced unique fragments not found with either MAV-1 or MAV-2 viral DNAs. The major viral component present in AMV-S stocks has the HindIII restriction pattern of MAV-1. Restriction enzyme analysis of the 5.3 X 10(6)-dalton unintegrated MAV viral DNAs and their integrated proviruses suggests that the DNAs have a direct terminal redundancy of approximately 0.3 megadaltons and integrate colinearly with respect to the unintegrated linear DNA.