Lyophilization, temperature control

The heat transfer system provides cooling required for freezing the product and the subsequent heat needed to establish rate of sublimation. Temperature control is required over the entire process, from the time the product is loaded onto the lyophilizer shelves until it is removed after stoppering. Therefore, cooling and... 

Searles, J.A., Carpenter, J.F., Randolph, T.W. The ice nucleation temperature determines the primary drying rate of lyophilization for samples frozen on a temperature-controlled shelf. PDAJ. Pharm. Sci. Technol. 90, 860-871, 2001... 

With continuous development of systems for controlled drug release, new materials are being used whose influence on peptide stability must be carefully examined. Thus, the model hexapeptide Val-Tyr-Pro-Asn-Gly-Ala (Fig. 6.30) embedded in poly (vinyl alcohol) and poly(vinyl pyrrolidone) matrices had rates of deamidation that increased with increasing water content or water activity, and, hence, with decreasing glass transition temperature (Tg). However, the degradation behavior in the two polymers differed so that chemical reactivity could not be predicted from water content, water activity, or T% alone. Furthermore, the hexapeptide was less stable in such hydrated polymeric matrices than in aqueous buffer or lyophilized polymer-free powders [132],... 

Over 50 different pyridazin-3-ones were evaluated for biological activity in a wheat (Triticum aestivum L.) test system described previously (1). Briefly, seeds were germinated in 9-cm petri dishes on three layers of filter paper. Pyridazinones were dissolved in acetone and the filter papers were impregnated with 1 ml of acetone solution. After the soluent evaporated, 10 ml of distilled water were added to form an inhibitor concentration of 100 yM. Seeds were planted directly on the moist papers and germinated for 4 days in a controlled environment chamber on a 16-hr photoperiod with 27+lC day temperature and 21+lC night temperature. Light intensity from both fluorescent and incandescent bulbs was 28 klux at dish level. Lipids were extracted and recovered from 1 g of lyophilized shoot tissue, separated into membrane and non-membrane lipids, and analyzed by gas chromatography as described (1). 

The PEG could stabilize proteins by two different temperature-dependent mechanisms. At lower temperatures, it is preferentially excluded from the protein surface but has been shown to interact with the unfolded form of the protein at higher temperatures, given its amphipathic nature (57). Thus, at lower temperatures, it may protect proteins via the mechanism of preferential exclusion, but at higher temperatures possibly by reducing the number of productive collisions between unfolded molecules. PEG is also a cryoprotectant and has been employed in Recombinate, a lyophilized formulation of recombinant Antihemophilic Factor, which utilizes PEG 3350 at a concentration of 1.5mg/mL. The low-molecular weight liquid PEGs (PEG 300-600) can be contaminated with peroxides and cause protein oxidation. If used, the peroxide content in the raw material must be minimized and controlled throughout its shelf life. The same holds true for polysorbates (discussed below). 

Shelf temperature uniformity across any one shelf and all of the shelves of the lyophilizer needs to be within an acceptable range to assure batch uniformity of the dried product. The temperature at any location is compared with either the mean of the measured values or the temperature indicated on the controlling instrument. The allowable range is dictated by the reference used, with tighter tolerances used when comparing the actual with the mean of the measurements. The stated capability for shelf temperature uniformity by many of the lyophilizer vendors is 1°C at steady-state conditions. Appropriately completed under no-load conditions, these functions may again be demonstrated under load conditions during the sublimation-condensation test. 

Alkali Treatment. The following is a typical procedure. A 1% solution of each protein in 0.1 N NaOH (pH< 12.5) was placed in a glass-stoppered Erlenmeyer flask and incubated at the appropriate temperature in a water bath. The final pH did not differ significantly from the initial value. After three hours, the sample was dialyzed against 0.01 N acetic acid for approximately two to three days and lyophilized. Control protein samples were dialyzed and lyophilized similarly. The pH was measured with a Coming pH meter before and after treatment. 

Stability of biopharmaceutical compounds should also be determined under conditions that mimic their normal usage. For instance, the stability of reconstituted lyophilized products should be assessed with respect to time and temperature and, if applicable, light and mechanical stimuli. Likewise, the stability of a compound included in implantable devices and controlled-release microsphere formulations should be determined over the course of its required use, under conditions which mimic the heat, moisture, light, and enzymatic physiological conditions to which it... 

Lyophilization (or freeze-drying) is a process utilized to convert a water-soluble material filled into a container to a solid state by removal of the liquid while frozen. The process requires the use of deep vacuums and careful control of temperatures. By conducting the process under reduced pressure, the water in the container converts from ice directly to vapor as heat is applied and is removed from the container by the vacuum. The dissolved solids in the formulation cannot undergo this phase change and remain in the container. At the completion of the cycle, the container will be returned to near atmospheric pressure stoppers are applied or fully seated and crimped as described above. Lyophilization is particularly common with biological materials whose stability in aqueous solution may be relatively poor. The time period in solution and the temperature of the solution are kept at a specified low temperature to prevent product degradation [35],... 

The remedy to this situation is to perform a thermal treatment of the frozen solution. This treatment consists of a controlled rewarming of the solution until devitrification and recrystallization of the excipient occurs, followed by a last freezing step below the solidification temperature. A typical excipient justifiable of such a treatment on thermodynamic grounds is mannitol. In the absence of proteins that maintain the structural integrity of the pellet, mannitol solutions often yield cakes of poor appearance. The induction of mannitol crystallization by rewarming around -25°C evades this problem and allows one to obtain elegant pellets that are easy to lyophilize and do not shrink. 

Lyophilization may be performed without pressure control, i.e., under maximum vacuum. In the absence of pressure control, the pressure generated in the lyophilizing chamber is sustained only by ice sublimation and/or water desorption. Under these conditions, the pressure is to be regarded as a dependent variable, as its actual level is dictated by other parameters such as shelf temperature, time, batch size, and product characteristics. 

With regard to process validation, it is essential to note that when the chamber pressure is controlled, the resultant supply of heat to the product and the progress of lyophilization depends on the combined effects of two intensive independent variables temperature and pressure. This is in contrast with the maximum vacuum process whereby shelf temperature is the only controllable intensive variable. 

Although pressure-controlled lyophilization brings significant advantages, a drawback of this technique is to risk promoting collapse in delicate formulations. This is because the assigned pressure modifies the course of sublimation and elevates product temperature in parts of the cycle where little evaporation would occur under maximum vacuum. 

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