Lyophilization cells

Molecular-Level Characterization of Lipid Bilayers in Disaccharide Matrices and Its Consequences for Cell Lyophilization... 

Viability of Lactobacillus acidophilus during storage at 37°C as a function of storage time. The cells were lyophilized in a solution of trehalose and phosphate ions. The symbols show results for cells lyophilized without additives (inverted triangles), with trehalose only (diamonds), with a trehalose-borate mixture (bullets), and with a trehalose-phosphate mixture (triangles). 

Nikolova and Ward [72,73] studied production of phenylacetyl carbinol from benz-aldehyde and pyruvate by whole-cell yeast biotransformation in two-phase systems. For the biocatalyst preparation fresh pressed commercial baker s yeast (50 g) was suspended in 50 ml 0.05 M sodium citrate buffer (pH 6.0) and lyophilized. Aliquots of 300 mg of lyophilized cells were mixed witii 1 g celite and the mixture was resuspended in 0.05 M sodium citrate buffer (pH 6.0). The suspension was lyophilized again and stored at 4°C. Scanning electron micrographs of the carrier celite and yeast cells lyophilized on celite are given in Fig. 1. Prior to use, organic solvents purchased in anhydrous form were saturated with 0,05 M sodium citrate buffer (pH 6.0). The same buffer was used as an aqueous component of the biphasic systems. 

Lymphoid cells Lymphomas Lymphosarcoma Lyochrome Lyophilization... 

Tank fermentation of Micromonospora inyoensis — Germination stage 1 Under aseptic conditions, add a lyophilized culture (or cells obtained from a slant culture) of M. inyoensis to a 300 ml shake flask containing 100 ml of the following sterile medium ... 

Scheme 9.3 Biooxidation of diols by lyophilized cells of R. ruber.

The microalgae are cultured in bioreactors under solar or artiflcial light in the presence of carbon dioxide and salts. The bioreactors may be closed systems made of polyethylene sleeves rather than open pools. Optimal conditions for pigment production are low to medium light intensity and medium temperatures (20 to 30°C). Pigment extraction is achieved by cell breakage, extraction into water or buffered solution, and centrifugation to separate out the filtrate. The filtrate may then be partly purified and sterilized by microfiltration and spray dried or lyophilized. 

Extraction procedures must be adjusted when separated anthocyanins will be tested in biological studies. We have found that the types of acids used for anthocyanin extraction as well as their residual concentrations in the final extract may affect the results obtained from biological tests. The growth inhibitory effect of anthocyanins on HT29 (human colonic cancer) cells may be overestimated if the residual acid in the extract exerts a toxic effect on the cells. Acetic acid residues in anthocyanin extracts showed less toxicity to HT29 cells than hydrochloric acid when samples were prepared under the same extraction procedure and subjected to the same tests on HT29 cells. In addition, the procedure to remove acids affected the acid residual concentration as well in final anthocyanin extracts, with lyophilization being more successful than rotary evaporation. 

Figure 3. SDS-PAGE and in situ pectinase activity on pectin and polygalacturonic acid-agarose overlays of culture filtrates of Aspergillus niger N-402 (upper panel) and Aspergillus FP-180 (lower panel) at 2.5, 3.5, 5.5 and 6.5 pHi (Lanes a, b, c, and d, respectively). Electrophoresis on 10% acrylamide slab gel (14 X 8 cm) in the presence of SDS was according to Laemmli (6), run at 30 mA constant current for 2 hours. Crude cell-free samples were concentrated by lyophilization, dialyzed, boiled with sample buffer by 60 sec. and applied to each well. Polyacrylamide gel and overlays were incubated overnight with 0.17 acetate buffer at room temperature.

J. Uy, M. Tanaka, T. Kishimoto, Y. Mass spectral analysis of complex lipids desorbed directly from lyophilized membranes and cells. Biochem. Biophys. Res. Comm. 1987,142,194-199. 

In another approach, the alcohol moiety, formed by an enzymatic hydrolysis of an ester, can act as a nucleophile. In their synthesis of pityol (8-37a), a pheromone of the elm bark beetle, Faber and coworkers [17] used an enzyme-triggered reaction of the diastereomeric mixture of ( )-epoxy ester 8-35 employing an immobilized enzyme preparation (Novo SP 409) or whole lyophilized cells of Rhodococcus erythro-polis NCIMB 11540 (Scheme 8.9). As an intermediate, the enantiopure alcohol 8-36 is formed via kinetic resolution as a mixture ofdiastereomers, which leads to the diastereomeric THF derivatives pityol (8-37a) and 8-37b as a separable mixture with a... 

Another very recent development in the field of enzymatic domino reactions is a biocatalytic hydrogen-transfer reduction of halo ketones into enantiopure epoxides, which has been developed by Faber, Bornscheuer and Kroutil. Interestingly, the reaction was carried out with whole lyophilized microbial cells at pH ca. 13. Investigations using isolated enzymes were not successful, as they lost their activity under these conditions [26]. 

The reduction of several ketones, which were transformed by the wild-type lyophilized cells of Rhodococcus ruber DSM 44541 with moderate stereoselectivity, was reinvestigated employing lyophilized cells of Escherichia coli containing the overexpressed alcohol dehydrogenase (ADH- A ) from Rhodococcus ruber DSM 44541. The recombinant whole-cell biocatalyst significantly increased the activity and enantioselectivity [41]. For example, the enantiomeric excess of (R)-2-chloro-l-phenylethanol increased from 43 to >99%. This study clearly demonstrated the advantages of the recombinant whole cell biocatalysts over the wild-type whole cells. 

KNOB protein, however, was shown to improve gene expression markedly (130-fold in HeLa cells). Additionally, it was shown that PEG could be conjugated to the surface of the nanospheres to prevent aggregation during lyophilization without a loss of bioactivity following one month in storage. The PEGylated particles, however, were cleared from mice at a slower rate than unmodified controls and were found to accumulate in the kidney and liver at 15 min after intravenous administration. There was no difference after one hour, however. 

After initial cell fermentation and product extraction from the producer cells, the crude preparation is subject to multiple chromatographic steps, including ion-exchange, hydrophobic interaction chromatography and gel-filtration chromatography. The purified product is presented in lyophilized form in vials (1 mg active/vial) and excipients include a phosphate buffer, sodium chloride and serum albumin. 

Neorecormon (tradename, also known as epoetin beta) is a recombinant human EPO first approved for medical use in the EU in 1997. It is indicated for the treatment of anaemia associated with various medical conditions, most commonly chronic renal failure and cancer patients receiving chemotherapy. Neorecormon is produced by recombinant DNA technology in a CHO cell line and is manufactured as outlined in Figure 10.5. It is presented in lyophilized format at various strengths (500-10 000 IU/vial) and contains phosphate buffer, sodium chloride, calcium chloride, urea, polysorbate and various amino acids as excipients. 

Keratinocyte growth factor is a 140 amino acid, 16.3 kDa member of the FGF family. It differs from native keratinocyte growth factor in that the first 23 N-terminal amino acids have been deleted, which improves its stability. After cell growth the product is recovered and purified by a multistep chromatographic protocol. It is presented in lyophilized format in single-use vials and containing mannitol, sucrose, polysorbate 20 and histidine as excipients. It is administered by daily i.v. injection, usually for several days. 

The wide-angle (201) reflection is strong, whereas the (200) reflection at 4.7 A Bragg spacing is much weaker or nonexistent for A8A and lyophilized HI, or is nearly as strong as the (201) reflection for SHal06-122 and solubilized/dried HI. Because the intersheet distance of polyalanine is —5 A, the unit cell should contain two //sheets. 

The popularity of this extraction method ebbs and flows as the years go by. SFE is typically used to extract nonpolar to moderately polar analytes from solid samples, especially in the environmental, food safety, and polymer sciences. The sample is placed in a special vessel and a supercritical gas such as CO2 is passed through the sample. The extracted analyte is then collected in solvent or on a sorbent. The advantages of this technique include better diffusivity and low viscosity of supercritical fluids, which allow more selective extractions. One recent application of SFE is the extraction of pesticide residues from honey [27]. In this research, liquid-liquid extraction with hexane/acetone was termed the conventional method. Honey was lyophilized and then mixed with acetone and acetonitrile in the SFE cell. Parameters such as temperature, pressure, and extraction time were optimized. The researchers found that SFE resulted in better precision (less than 6% RSD), less solvent consumption, less sample handling, and a faster extraction than the liquid-liquid method [27]. 

---