Lyophilized protein materials

Freeze drying, or lyophilization, is normally reserved for temperature-sensitive materials such as vaccines, enzymes, microorganisms, and therapeutic proteins, as it can account for a significant portion of... 

The presence of a solvent, especially water, and/or other additives or impurities, often in nonstoichiometric proportions, may modify the physical properties of a solid, often through impurity defects, through changes in crystal habit (shape) or by lowering the glass transition temperature of an amorphous solid. The effects of water on the solid-state stability of proteins and peptides and the removal of water by lyophilization to produce materials of certain crystallinity are of great practical importance although still imperfectly understood. 

Molecular imprinting is not limited to organic polymer matrices, but can also be applied to silica-based materials and even proteins. Proteins freeze-dried in the presence of a transition state analogue as template have been used successfully as catalysts, e.g., for the dehydrofluorination of a fluorobutanone. For instance, lyophilized 3-lactoglobulin imprinted in this manner with N-isopropyl-N-ni-trobenzyl-amine could accelerate the dehydrofluorination by a factor of 3.27 compared to the non-imprinted protein see Table 5 [62]. In a similar procedure, BSA was imprinted with N-methyl-N-(4-nitrobenzyl)-S-aminovaleric acid and showed an enhancement of the catalytic effect by a factor of 3.3 compared to the control protein for the same reaction see Table 5 [113]. 

Although most assays perform well with regard to specificity and reproducibility, the major problem remains their standardization (A9, Dl, K30, L4). There is currently no internationally accepted standard, and the selection of a reference material raised many problems (A8, G5, K30, L4). A number of questions have not been solved Should the standard consist of several apo(a) isoforms Can the reference material be lyophilized Should results be expressed as mass or as moles of apoprotein or lipoprotein How should the protein mass of the primary standard be determined What are optimal storage conditions for the secondary standard Which method can be used as a reference method Can recombinant apo(a) represent an alternative for a primary standard These problems came to light in the course of the international surveys whose results were presented at the Lp(a) Workshop in New Orleans (1992) (L4). 

Material. 3 X crystallized, dialyzed and lyophilized Grade 1 Hen Egg White Lysoz3mie was obtained from Sigma Chemical Company, and used without further purification. Purity of the protein was verified by both gel electrophoresis on acrylamide and chromatography on a column packed with anion exchange resin (Cl form) Sephadex DEAE. The sample showed a single peak in these analyses. 

Dialysis and ultraflltration have been largely applied to isolate and fractionate food proteins and peptides. To isolate the protein fraction from wine and must samples, different authors used dialysis followed by lyophilization to concentrate the dialyzed samples [106,108,109], Depending on the application, membrane of different material, filtration surface and cut-off, able to fractionate the molecules in function of their molecular size, can be used to remove either proteins and other macromolecules or amino acids and small peptides. 

Lyophilization is a process in which a solution of a drug is frozen to a solid and the solvent, usually water, removed by sublimation on exposure to a vacuum. The process has been studied intensively because it can be applied to the preservation of labile drugs or materials such as proteins which would otherwise be adversely affected by the solvent over a period of time. 

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). 

The classical use of the term lyophilic colloids refers to soluble macromolecular materials in which the individual particles (macromolecules such as synthetic polymer chains or proteins)... 

Decades ago, biochemists recognized that it was difficult to remove all water from a large number of macromolecular materials. The term bound water was coined to explain the great affinity many of these materials showed for water, particularly the proteins. Biochemists were convinced that biological behavior, at least in part, resulted from the amount of bound water contained in the macromolecular structure. As an example, water held in plant structures so that it did not freeze in below-freezing temperatures was considered to be bound. At the time these ideas found favor, the method of lyophilization or quick freeze-drying had not been... 

Wash, hand-peel, chop and lyophilize each plant tissue material for 3-4 h at 25°C (e.g., soybean (Table 17.1), potato, banana, eggplant, sweet potato and artichoke (Table 17.2). Grind the dry tissue to obtain a fine powder and select the particle size using sieves of lower than 200 pm mesh. Store the dry powder in a desiccator at 25°C and use it as the enzymatic source of PPO or peroxidase in the preparation of biosensor. Determine enzymatic activity and total protein content as described in Procedure 22 (in CD accompanying this book). Prepare the carbon paste electrode with dry tissue as described in the same procedure. 

In the past few years great technical progress has been made in lyophiliza-tion. Lyophilization and ampouling under nitrogen also overcomes the problems of destructive postal delays. Therefore, the BCR has decided to prepare the reference material as lyophilized human serum. Some people believe that lyophilization alters the properties of the serum to such an extent that problems can arise in matrix-dependent measuring techniques such as radioimmunoassay (RIA) and competitive protein binding (CPB) (Menson and Adams, 1977 Fraser et al., 1978 DiSilvio, 1979). Indeed, before the lyophilized material can be used it should be checked to determine whether the material can be satisfactorily reconstituted and whether it can be assayed by the different techniques currently in use in clinical laboratories. 

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