A recombinant lipoprotein antigen against Lyme disease expressed in E. coli: fermentor operating strategies for improved yield.

Decorin-binding lipoprotein, lpp-DBP, a bacterial surface adhesin, shows promise as a vaccine against Lyme disease. It is expressed in recombinant E. coli as an undesirable 20.5 KDa apoprotein that is subsequently lipidated in vivo to the desired 22 KDa lpp-DBP form. This study defines fermentation conditions for maximizing lpp-DBP yield. Super broth medium, a low post-induction temperature (30 degrees C), and a glucose feed based on dissolved oxygen resulted in high lpp-DBP yield and minimized apoprotein formation. Since cells lysed within 2-3 h after induction, the cell yield was maximized by growing cells to high cell density prior to induction. Compared to a glucose feed based on maintaining a constant fermentor glucose concentration (Glucose-Stat), feeding based on maintaining a constant dissolved oxygen level (DO-Stat) improved yields. Also, a dissolved oxygen level of 60% (air saturation) was best, as no product degradation was detected by Western blotting and SDS-PAGE. Acetic acid levels under both modes of glucose feed were sufficiently low, and no adverse growth effects were observed.

Comparability testing of a humanized monoclonal antibody (Synagis) to support cell line stability, process validation, and scale-up for manufacturing.

Biochemical and functional testing of a humanized monoclonal antibody directed against Respiratory Syncytial Virus (Synagis) has been performed to evaluate cell line stability, support process validation, and to demonstrate "comparability" during the course of process development. Using a variety of analytical methods, product manufactured at different sites and in bioreactors from 20 litres to 10,000 litres was shown to be biochemically and functionally equivalent. The biochemical testing for microheterogeneity found on Synagis included evaluation of changes in post-translational modifications such as deamidation, truncation, and carbohydrate structure. Studies were also performed to support cell line stability assessment and cell culture process validation. Cell culture conditions were deliberately varied in an attempt to determine if this would have an impact on the microheterogeneity of the product. In these studies Synagis was produced from cells cultured beyond the population doublings achieved at the maximum manufacturing scale, under conditions of low glucose, and using harvest times outside of the historical manufacturing operating range. Results showed that there was a different pattern of glycosylation during the early stages of bioreactor culture. No other changes in microheterogeneity were apparent for the other culture conditions studied. In summary, comparability assessment demonstrated that the Synagis manufacturing process is robust and consistent resulting in a predictable and reproducible monoclonal antibody product.

Production of parvovirus B19 vaccine in insect cells co-infected with double baculoviruses.

Recombinant human parvovirus B19 virus-like particles (VLPs), a candidate vaccine, were produced using the insect cell (Sf-9)-baculovirus (AcNPV) expression system. The synthesis and assembly of the particles in Sf-9 cells are directed by double infections with one recombinant virus (bacVP1) expressing the parvovirus minor viral protein VP1 and a second virus (bacVP2) expressing the major viral protein VP2. Previous animal studies demonstrated that the polypeptide composition of the VLPs strongly affects the elicitation of virus neutralizing antibodies. The key factor controlling the production of an immunologically potent product in bioreactors was identified to be the multiplicity of infection (MOI) of bacVP1 and bacVP2 used for infection. A probabilistic model, which correlates well with the experimental results, was employed to facilitate the selection of MOIs and to provide a better understanding of the baculovirus co-infection process. A novel production process based on secondary infections was developed to ensure product consistency and to simplify large-scale logistics. The effects of other critical process parameters, such as temperature, dissolved oxygen concentration, lactate concentration, cell concentration at infection, and harvest time, were also investigated. (c) 1996 John Wiley & Sons, Inc.

Effects of heat shock on the production of human erythropoietin from recombinant CHO cells.

The production of recombinant proteins by mammalian cells demands a highly controlled environment for cell cultivation. Temperature stress represents a commonly encountered disturbance in both research and process environments. In this study, we examined the effects of heat shock on the expression of recombinant human erythropoietin (EPO) in a Chinese hamster ovary (CHO) cell line. Biosynthetic radiolabeling experiments indicated that the cells exposed to a 42 degrees C/1-hour heat shock exhibit a transient reprogramming of transcription and translation characterized by the inhibition of protein synthesis and induction of heat shock proteins. The rate of protein synthesis decreased by 50% after the heat shock, while the rate of RNA synthesis increased by 50% initially and then quickly reduced to 80% of the control level. The protein and RNA synthesis rates were fully recovered in approximately 48 hours after the heat shock. However, we found that the expression of EPO was not arrested by the heat shock. The glycosylation patterns, as examined by isoelectric focusing, of either the culture supernatant or the purified EPO were not affected by the heat shock. In contrast, a 45 degrees C/1-hour heat shock terminated RNA and protein synthesis immediately and caused culture death in 12 hours. Cellular responses to temperature stress were affected by the metabolic state of the cells; cells maintained in serum-free medium were more sensitive than cells growing exponentially in the presence of serum. We have also examined the kinetics of metabolic responses of the cells to heat shock with respect to nutrient uptake and metabolite accumulation.