Cell freeze drying

Fresh A,W,D A, I, W, D With cells (freeze-dried) Separated (from cells) With (medium)... 

Log-phase cells were subjected to a 350nM pulse of [1- C]acetate, (for acyl-CoA pool analyses [ C-acetate] = 700nM) for 30 mins, after which the pulse was diluted 100 fold with unlabelled acetate. Samples were removed from the vessel, the cells freeze-dried and lipids extracted and analysed as above. 

The harvest is a very eomplex mixture of bacterial cells, metabolic products and exhausted medium. In the ease of a live attenuated vaccine it is innocuous and all that is necessary is for the baeteria to be separated and resuspended in an appropriate menstmum, possibly for freeze-drying. In a vaccine made Irom a pathogen the harvest may be intensely dangerous and great care is necessary in the following procedures. 

BCG Cultures of live BCG cells in liquid or on solid media 1 Bacteria centrifuged from medium 2 Resuspension in stabilizer 3 Freeze-drying Viable count induction of sensitivity to tuberculin in guinea-pigs Exclusion of virulent mycobacteria absence of excessive dermal reactivity... 

Measles Chick embryo cell cultures infected with attenuated measles virus 1 Clarification 2 Freeze-drying Infectivity titration in cell cultures Tests to exclude presence of extraneous viruses... 

To prepare the mild-alkali-extract, dry watermelon cell walls were suspended in a solution of 0.1 N NaOH, and allowed to react with stirring at room temperature for 15 minutes. A pH of 13, as indicated by pH paper, was kept constant during this period by addition of 0.1 N NaOH. To ensure complete reaction, the treatment was continued overnight at 4 °C. The soluble portion was separated by centrifugation at 10,000 RPM for 20 minutes in a Sorval GSA rotor. The insoluble portion was washed twice with water. The supernatants were combined and, after neutralization to pH 7.0 with acetic acid, dialyzed against distilled water and freeze dried. 

The dimer chains of Ca -ATPase can also be observed by freeze-fracture electron microscopy [119,165,166,172-174], forming regular arrays of oblique parallel ridges on the concave P fracture faces of the membrane, with complementary grooves or furrows on the convex E fracture faces. Resolution of the surface projections of individual Ca -ATPase molecules within the crystalline arrays has also been achieved on freeze-dried rotary shadowed preparations of vanadate treated rabbit sarcoplasmic reticulum [163,166,173,175]. The unit cell dimensions derived from these preparations are a = 6.5 nm b = 10.7 nm and 7 = 85.5° [175], in reasonable agreement with earlier estimates on negatively stained preparations [88]. 

The effect of oil/water ratio has been studied extensively for various catalysts. Patel et al. [258] reported effect of oil/water ratio on rate of desulfurization by IGTS8. They used freeze-dried cells reconstituted with water to do the studies. They found that a minimum of 1.25 mL of water per gram of freeze-dried cells is necessary to enable biodesulfurization. At a W/O ratio of 1 9, about 82% of the maximum desulfurization activity was achieved. The rate of desulfurization was reported to be similar between the W/O ratio of 1 1 and 4 1, but decreased upon increasing oil content further. The effect of the ratio was also studied by Shan et al. using diesel oil as the oil phase and P. delafieldii R-8 as the biocatalyst [259], The water content was varied to obtain a W/O ratio between 0 1 and 20 1, using a fixed amount of biocatalyst and oil. The authors found that the desulfurization rate increased up to a W/O ratio of 2 1, after which it remained constant. 

Neumega is the tradename given to the IL-ll-based product approved for the prevention of thrombocytopenia. The product is produced in engineered E. coli cells and is presented as a purified product in freeze-dried format. Excipients include phosphate buffer salts and glycine. It is reconstituted (with water for injections) to a concentration of 5 mg ml-1 before s.c. administration. 

Enbrel is a product now approved for medical use that is based upon this strategy. The product is an engineered hybrid protein consisting of the extracellular domain of the TNF p75 receptor fused directly to the Fc (constant) region of human IgG (see Box 13.2 for a discussion of antibody structure) The product is expressed in a CHO cell line from which it is excreted as a dimeric soluble protein of approximately 150 kDa. After purification and excipient addition (mannitol, sucrose and trometamol), the product is freeze-dried. It is indicated for the treatment of rheumatoid arthritis and is usually administered as a twice-weekly s.c. injection of 25 mg product reconstituted in WFI. Enbrel functions as a competitive inhibitor of TNF, a major pro-inflammatory cytokine. Binding of TNF to Enbrel prevents it from binding to its true cell surface receptors. The antibody Fc component of the hybrid protein confers an extended serum half-life on the product, increasing it by fivefold relative to the soluble TNF receptor portion alone. 

After its purification, sterile filtration and aseptic filling, human urokinase is normally freeze-dried. Because of its heat stability, the final product may also be heated to 60 °C for up to 10 h in an effort to inactivate any undetected viral particles present. The product utilized clinically contains both molecular mass forms, with the higher molecular mass moiety predominating. Urokinase can also be produced by techniques of animal cell culture utilizing human kidney cells or by recombinant DNA technology. 

Cerezyme is produced in a CHO cell line harbouring the cDNA coding for human (i-glucoccr-ebrosidase. The purified product is presented as a freeze-dried powder, which also contains mannitol, sodium citrate, citric acid and polysorbate 80 as excipients. It exhibits a shelf life of 2 years when stored at 2-8 °C. 

Figure 13.5 Outline of the production strategy of CEA-SCAN. The antibody-producing hybridoma cell line was originally obtained by standard methods of hybridoma generation. Spleen-derived murine B-lymphocytes were fused with murine myeloma calls. The resulting stable hybridomas were screened for the production of anti-CEA monoclonals. The clone chosen produces an IgG anti-CEA antibody. Note that the finished product outlined above is not radiolabelled. The freeze-dried antibody preparation (which has a shelf life of 2 years at 2-8 °C) is reconstituted immediately prior to its medical use. The reconstituting solution contains 99mTc, and is formulated to facilitate direct conjugation of the radiolabel to the antibody fragment...

Hsu et al. [1.121] observed recrystallization on the recombinant CD4-IgG with a cryomicroscope cooled to -60 °C by a cascade of four Peltier modules. The observation cell can also be evacuated for freeze drying studies. 

---