Denaturation freeze-drying

Hanafusa, N. (1969). Denaturation of enzyme proteins by freeze drying. In Freezing and Drying of Microorganisms, ed. T. Nei, pp. 117-29. Baltimore, MD University Park Press. 

The coacervation approach uses heating or chemical denaturation and desolvation of natural proteins or carbohydrates. As much as 85% of water-soluble drugs can be entrapped within a protein matrix by freeze-drying the emulsion prepared in this manner. For water-insoluble drugs, a microsuspension-emul-sion procedure has been suggested as a method of choice to achieve high drug payloads. 

Native, multi-subunit KLH also should not be frozen. Freeze-thaw effects cause extensive denaturation and result in considerable amounts of insoluble material. Commercial preparations of native KLH are typically freeze-dried solids that no longer fully dissolve in aqueous buffers and do not display the protein s typical blue color due to loss of chelated copper. The partial denatured state of these products often makes conjugation reactions difficult. 

Figure 8.7 Overview of the manufacture of Betaferon, a recombinant human IFN-(3 produced in E. coli. The product differs from native human IFN-(3 in that it is unglycosylated and cysteine residue 17 had been replaced by a serine residue. E. coli fermentation is achieved using minimal salts/glucose media and product accumulates intracellularly in inclusion body (IB) form. During downstream processing, the lbs are solubilized in butanol, with subseguent removal of this denaturant to facilitate product refolding. After two consecutive gel-filtration steps, excipients are added, the product is filled into glass vials and freeze-dried. It exhibits a shelf life of 18 months when stored at 2-8 °C...

Deactivation due to lyophilization by non-reversible denaturation in freeze/dry cycle... 

Despite some conflicting evidence (Kinsella and Fox, 1986), it appears that denaturation has little influence on the amount of water bound by whey proteins. However, other factors which may accompany denaturation (e.g. Maillard browning, association or aggregation of proteins) may alter protein sorption behaviour. Drying technique affects the water sorption characteristics of WPC. Freeze-dried and spray-dried WPC preparations bind more water at the monolayer level than do roller-, air- or vacuum-dried samples, apparently due to larger surface areas in the former. As discussed above, temperature also influences water sorption by whey protein preparations. The sorption isotherm for /Mactoglobulin is typical of many globular proteins. 

For high-performance lAC, the preferred solid support is a glass bead solid support coated wilh either protein-A or protein-A covalently linked with the antibody through a carbodiimide bond (165, 166). In either case, protein-A binds to the Fc portion of the antibody so that the combining sites are oriented to the mobile phase. Once the protein is attached, the lAC matrix is packed into the column either as a slurry or dry. Pump-slurry techniques use buffers with a low salt content, such as Tris or 0.01 M phosphate buffer to minimize friction and denaturation of the immobilized antibody (16). If the solid support consists of glass beads, the packing can be freeze-dried after antibody attachment and packed dry. 

As with any laboratory method, there are precautions and limitations of lyophilization that must be understood. Only aqueous solutions should be lyophilized. Organic solvents lower the melting point of aqueous solutions and increase the chances that the sample will melt and become denatured during freeze-drying. There is also the possibility that organic vapors will pass through the cold trap into the vacuum pump, where they may cause damage. 

Some freeze-dried antisera are difficult to reconstitute, or occasionally may lose activity. Test a small sample before drying the whole batch. Any cloudiness after reconstitution is denatured lipoprotein, and can be clarified by centrifugation and does not affect antibody binding. 

Samples used for analyses of functional properties were the redissolved protein fractions (RDP) from the unheated, the 60 sec microwave treatment and from the hot water treatments that had subsequently been freeze-dried, a procedure reported to result in minimum denaturation (7.) - At least six determinations were done for each test. 

Probiotics may be encapsulated in protein-based emulsions. Picot and Lacroix (2003) prepared microcapsules by emulsifying milkfat containing micronized skim milk powder (as a surrogate for freeze-dried bacteria) with heat denatured whey proteins and then spray drying. Incorporation rates of up to 58% milk fat and 29% skim milk... 

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