Freeze drying reconstitution

The use of UF and RO membranes as a concentration procedure (compared to other concentration techniques for DOP such as freeze-concentration, freeze-drying-reconstitution, anion-exchange-small-volume elutions,... 

Lyophilization. LyophiLization is essentially a drying technology. Some dmgs and biologicals are thermolabile and/or unstable in aqueous solution. Utilization of freeze drying permits the production of granules or powders that can be reconstituted by the addition of water, buffered solution, or mixed hydrophilic solvents just prior to use, eg, certain antibiotic suspensions. 

Each interferon preparation was ultracentrifuged at 20,000 revolutions per minute for one hour to remove tissue debris and inactivated virus. The supernatant was dialyzed against distilled water (1 400) for 24 hours at4°C. The material was then freeze-dried. The dried product was reconstituted in one-tenth of the original volume in distilled water and dispensed into ampoules. Reconstituted solutions were assayed for interferon activity, examined for toxicity, and tested for sterility. 

Bone defects surgically produced in sheep and rabbit models, have been treated with freeze dried methylpyrrohdinone chitosan [334-336]. hi view of improving bone tissue reconstitution with chitosan associated with calcium phosphate. Microscopic and histological analyses showed the presence of an osteogenic reaction moving from the rim of the surgical lesion toward the center. In control lesions, dense fibrous tissue, without the characteristic histoarchitecture of bone was observed. 

Dry powders for reconstitution as an injectable product may be produced by several methods filling the product into vials as a liquid and freeze-drying, aseptic crystallization followed by powder filling, and spray-drying followed by powder filling. A brief discussion of each follows. 

Most proteins are not sufficiently stable in aqueous solution to allow formulation as a sterile solution. Instead, the protein is freeze-dried and reconstituted before use. Development of a freeze-dried protein formulation often requires special attention to the details of the freezing process (potential pH shifts and ionic strength increase with freezing) as well as to potential loss of activity with drying. Formulation additives, such as sugars and polyhydroxy compounds, are often useful as cryoprotectants and lyoprotectants. Residual moisture may also be critical to the stability of the dried preparation [33],... 

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. 

The F/P ratio of the purified, labeled protein may be determined by measuring the absorbance at 345 and 280nm. Ratios between 0.3 and 0.8 usually produce labeled molecules having acceptable levels of fluorescent intensity and good retention of protein activity. AMCA-labeled proteins may be lyophilized without significant loss of fluorescence. The addition of bovine serum albumin (15mg/ml) or another such stabilizer is often necessary to retain solubility of the freeze-dried, labeled protein after reconstitution. 

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. 

Streptokinase is a 48 kDa extracellular bacterial protein produced by several strains of Streptococcus haemolyticus group C. Its ability to induce lysis of blood clots was first demonstrated in 1933. Early therapeutic preparations administered to patients often caused immunological and other complications, usually prompted by impurities present in these products. Chromatographic purification (particularly using gel filtration and ion-exchange columns) overcame many of these initial difficulties. Modern chromatographically pure streptokinase preparations are usually supplied in freeze-dried form. These preparations (still obtained by non-recombinant means) often contain albumin as an excipient. The albumin prevents flocculation of the active ingredient upon its reconstitution. 

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...

Gu and Gao [3.42] reported that freeze dried cyclophosphamide in liposomes (CPL) reconstitutes well and has a larger antitumor activity and a smaller toxitity than CPL in aqueous solution. 

Fig. 3.21. Retention rate of liposome-encapsulated hemoglobin as a function of the time elapsed after the reconstitution of the freeze dried LEH, with different trehalose concentrations as CPA. 1, no trehalose 2, 10 mM 3, 50 mM 4, 150 mM 5, 300 mM trehalose (Fig. 2 from [3.43]).

Medas, M Simatose, D. Freeze-drying and reconstitution of raspberries, influence of the chemical content and variety. International Institute of Refrigeration (XIII Congress, p. 605-610, Washington, 1971... 

The most important goal of freeze-drying is to produce a substance with good shelf stability and which is unchanged after reconstitution with water, though this depends also very much on the third step of the process the packing and conditions of storage. 

Preparation of cotton bract extracts. Figure 1 is a flow chart showing our procedures for preparing the various bract extracts. Dried bracts (frost killed) were hand picked just prior to harvest from cotton fields in the Lubbock, Texas area. These were stored at room temperature. Extracts were freeze-dried and stored at -4°C. For inhalation challenge by our subjects each extract was reconstituted with water or saline, as indicated, at a concentration equivalent to the standard crude extract. This Insured that for challenge purposes components were not concentrated as purification progressed. 

Practically all available iodinated extracellular X-ray contrast agents have been encapsulated into liposomes using different lipids and methods of preparation. Table 1 gives a short and intentionally incomplete overview of some of the approaches. The first liposomal contrast agent preparation that was tested in humans contained diatrizoate [48]. The injected dose was up to 0.5 ml kg k The preparation was effective even in plain radiography where lesions down to 0.8-1.0 cm could be detected in patients. However, adverse events such as fever and hyperthermia, which occurred in 30% of the patients, limited further use. We have incorporated iopromide into MLVs that were prepared from phosphatidyl choline (PC), cholesterol and stearic acid at a molar ratio of 4 5 1 using the ethanol-evaporation technique [44]. The liposomes can be stored freeze-dried and they are reconstituted before use by... 

IV injection (20 or 40 mg) over no less than 3 minutes Reconstitute the freeze-dried powder with 5 ml of 0.9% sodium chloride injection. Withdraw 5 ml of the reconstituted solution and administer as an IV injection over no less than 3 minutes. 

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