Lyophilization proteins

B. S. Chang and C. S. Randall, Use of subambient thermal analysis to optimize protein lyophilization. Cryobiology 29 632-656 (1992). 

The final formulation is achieved by concentration and diafiltration into a citrate, glycine, sodium chloride buffer with the proper ionic strength and dilution to the final protein concentration of approximately 25 mg mL . The product is filled into vials (10 mL, containing -250 mg protein), lyophilized, and then treated in a validated terminal viral inactivation step (Step 6). 

R Johnson, C Kirchhoff, H Gaud. Mannitol-sucrose mixtures - versatile formulations for protein lyophilization. Journal of Pharmaceutical Sciences 91 914-922, 2002. 

Passot, S., Fonseca, F., Trelea, I. C., Remillieux, A., Galan, M., Morris, G. J Marin, M., 2007. Controlled nucleation improves efficiency of pharmaceutical protein lyophilization. Proceedings of the Joint Conference of AFSIA and Drying Working Group of EFCE, Biarritz, France, 54-55. 

TABLE 20 Sources of Aggregation Specific to Protein Lyophilization... 

Detailed studies of the apolipoproteins require the removal of the lipid from the lipoproteins. This can be achieved by extraction of the lipoproteins either direct from plasma or in the precipitated form with ethanol-diethyl ether mixtures (3 1, v/v). The more powerful lipid solvent, chloroform-methanol (2 1, v/v) results in a poorly soluble apolipoprotein. With ethanol-ether, however the advantage of having a soluble apoprotein is partly offset by the loss of 20% of VLDL protein. Lyophilization before... 

Rate effects may not be chemical kinetic ones. Benson and co-worker [84], in a study of the rate of adsorption of water on lyophilized proteins, comment that the empirical rates of adsorption were very markedly complicated by the fact that the samples were appreciably heated by the heat evolved on adsorption. In fact, it appeared that the actual adsorption rates were very fast and that the time dependence of the adsorbate pressure above the adsorbent was simply due to the time variation of the temperature of the sample as it cooled after the initial heating when adsorbate was first introduced. 

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... 

Sterile agar slants are prepared using the Streptomyces sporulation medium of Hickey and Tresner, J. Bact., vol. 64, pages 891-892 (1952). Four of these slants are inoculated with lyophilized spores of Streptomyces antibioticus NRRL 3238, incubated at 28°C for 7 days or until aerial spore growth is well-advanced, and then stored at 5°C. The spores from the four slants are suspended in 40 ml of 0.1% sterile sodium heptadecyl sulfate solution. A nutrient medium having the following composition is then prepared 2.0% glucose monohydrate 1.0% soybean meal, solvent extracted, 44% protein 0.5% animal peptone (Wilson s protopeptone 159) 0.2% ammonium chloride 0.5% sodium chloride 0.25% calcium carbonate and water to make 100%. 

However, in most cases enzymes show lower activity in organic media than in water. This behavior has been ascribed to different causes such as diffusional limitations, high saturating substrate concentrations, restricted protein flexibility, low stabilization of the enzyme-substrate intermediate, partial enzyme denaturation by lyophilization that becomes irreversible in anhydrous organic media, and, last but not least, nonoptimal hydration of the biocatalyst [12d]. Numerous methods have been developed to activate enzymes for optimal use in organic media [13]. 

The simplest way to prepare a biocatalyst for use in organic solvents and, at the same time, to adjust key parameters, such as pH, is its lyophilization or precipitation from aqueous solutions. These preparations, however, can undergo substrate diffusion limitations or prevent enzyme-substrate interaction because of protein-protein stacking. Enzyme lyophilization in the presence of lyoprotectants (polyethylene glycol, various sugars), ligands, and salts have often yielded preparations that are markedly more active than those obtained in the absence of additives [19]. Besides that, the addition of these ligands can also affect enzyme selectivity as follows. 

Gel Filtration. The lyophilized protein was redissolved in 50 mM phosphate buffer, pH 7.4 0.15 m NaCl 0.013 % sodium azide and loaded on a Superdex 75HR1030 column equilibrated with the same buffer. Elution was downward flow (0.15 ml/min) and 0.25 ml fi actions were collected. Fractions with pectin lyase activity were combined, dialyzed against distilled water and used in the next step. To estimate the molecular mass of PNL, the column was calibrated with standard proteins (Sigma MW-GF-70 Albumin, 66,000 Da Carbonic Anhidrase, 29,00 Cytochrome, 12,400 and Aprotinin, 6,500). The proteins were eluted in the conditions described above and their volumes (F ) were calculated fi om the peak maximum of the absorbance at 280 nm. The partition coefficient was obtained fi om the relationship where F, represents the bed volmne of column and F the void volume (which was calculated using blue dextran. Sigma). The molecular mass was determined using a standard curve of vs the logarithm of the molecular masses of the standards [28, 29]... 

Exudate collection in trap solutions usually requires subsequent concentration steps (vacuum evaporation, lyophilization) due to the low concentration of exudate compounds. Depending on the composition of the trap solution, the reduction of sample volume can lead to high salt concentrations, which may interfere with subsequent analysis or may even cause irreversible precipitation of certain exudate compounds (e.g., Ca-citrate, Ca-oxalate, proteins). Therefore, if possible, removal of interfering salts by use of ion exchange resins prior to sample concentration is recommended. Alternatively, solid-phase extraction techniques may be employed for enrichment of exudate compounds from the diluted trap solution (11,22). High-molecular-weight compounds may be concentrated by precipitation with organic solvents [methanol, ethanol, acetone 80% (v/v) for polysaccharides and proteins] or acidification [trichloroacetic acid 10% (w/v), per-... 

Extraction of lyophilized protein powder by w/o-ME solution (solid-phase extraction method, SPE). 

Elumatrope (human growth hormone) Lyophile for IV injection, recon. with 0.3% m-cresol, 1.7% glycerin Stable for 14 days at 2-8°C 5 mg protein per vial, 1.13 mg Na2HP04, 25 mg glycine, 25 mg mannitol... 

Roferon (interferon a-2a) Solution or lyophile for IV injection Stable for 1 month at 2-8°C after reconstitution Each mL contains 3, 6, 36 million IU protein with 9 mg NaCl, 5 mg HAS, and 3 mg phenol... 

The degradation of proteins in the solid state occur to a lesser extent and typically via different mechanisms than those that occur in solution [109,110]. Lyophilization is currently the more common technique in the manufacture of dried therapeutic proteins however, there is increasing interest in the use of spraydrying, owing to the unique physical nature of the spray-dried powder and its potential usefulness in protein drug delivery. 

Stabilization of proteins against those degradative processes with retention of structure and function through removal of water requires an understanding of the process of lyophilization or freeze-drying. 

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