Lyoprotectant

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. 

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],... 

M. Ressing, W. Jiskoot, H. Talsma, C. van Ingen, E. Beuvery, and D. Crommelin, The influence of sucrose, dextran, and hydroxypropyl-beta-cyclodextrin as lyoprotectants for freeze-dried mouse IgG2a monoclonal antibosy (MN12), Pharm. Res., 9, 266... 

Townsend and De Luca have studied the influence of lyoprotectans (LP) on ribonu-clease (Ri) [3.14-3.17] as a protein model. Lyoprotection is defined as stabilization and prevention of degeneration of macromolecules during freeze drying as well as during storage. With phosphate buffer at pH 3 to pH 10, Ri in the dry stage loses its activity at 45 °C... 

Studies with the freeze dried DPPC liposomes in trehalose solution showed, that not Tg )f the amorphous sugar is the critical temperature during storage, but the bilayer transition emperature Tm. for the lyposomes determines the short term stability of the formulation. With trehalose as lyoprotectant and a low residual water content, Tm proved to be 10 to 30 °C below the onset of T . 30 min heating above Tm but well below T% decreased the retention of CF after rehydration. Tm< after the heating was reduced from 40 to 80 °C to below 25 °C. 

Lactose, trehalose and maltose have equally lyoprotectant properties, while liposomes with sucrose showed an increase in size. 

Most enzyme powders are prepared by lyophilisation (freeze drying). However, the lyophilization procedure might inactivate the enzyme to some extent. To avoid this and thereby increase the activity of lyophilized enzymes in dry organic solvents, the lyophilization can be carried out in the presence of lyoprotectants such as sorbitol (Dabulis and Klibanov, 1993). The inactivation is believed to be caused at least partly by a reversible conformational change in the enzyme. This process can be reversed and the enzyme reactivated by the addition of small amoimts of water (Dabulis and Klibanov, 1993). 

Dabulis, K. and Klibanov, A.M. (1993) Dramatic enhancement of enzymatic activity in organic solvents by lyoprotectants. Biotechnol. Bioeng., 41, 566-571. 

Lyoprotectants can affect enzyme stability in both stages of lyophilization the freezing and the drying stages. In the freezing stage of lyophilization, ice crystals form and have been shown to be a cause of enzyme denaturation. Studies have shown that when added as a lyoprotectant, the amorphous polyol mannitol stabi-... 

When 18-crown-6 was co-lyophilized with a-chymotrypsin, a 470-fold activation was seen over the free enzyme in the transesterification of APEE with 1-propanol in cyclohexane (Scheme 3.2) [96]. There was a low apparent specificity for the size and macrocyclic nature of the crown ether additives, suggesting that, during lyophilization, 18-crown-6 protects the overall native conformation and acts as a lyoprotectant. To examine this global effect, FTIR was used to examine the effect of crown ethers on the secondary structure of enzymes. In one study [98], subtilisin Carlsberg was shown to retain its secondary structure in 1,4-dioxane when lyophi-lized in a 1 1 ratio with 18-crown-6. In addition, examination of FTIR spectra from varying incubation temperatures indicated that an increase in crown ether content in the final enzyme preparation resulted in a decreased denaturation temperature in the solvent, indicating a more flexible protein structure. 

Tonicity agent/stabilizer Provides isotonicity to the formulation such that it is suitable for injection Examples include polyols, salts, and amino acids Help maintain the protein in a more compact state (polyols) Minimize electrostatic, solution protein-protein interactions (salts) Stabilizers include cryo and lyoprotectants Examples include polyols, sugars, and polymers Cryoprotectants protect proteins from freezing stresses Lyoprotectants stabilize proteins in the freeze-dried state... 

Of course, it is common (and often desireable) to have both amorphous and crystalline phases present in a freeze-dried formulation. This is particularly relevant to freeze-dried proteins, where the lyoprotectant is present in the amorphous state, and another component, such as glycine or mannitol, is present as a crystalline solid in order to impart mechanical integrity and pharmaceutical elegance to the lyophilized solid. 

To protect the active constituent during freeze-drying (lyoprotectants). 

Van Winden et al. [1.161] used MTDSC in lyoprotected liposomes to detect the glass transition in samples in which it overlaps with the bilayer melting endotherm. 

Raman spectroscopy was used by Sane et al. [1.165] to quantitate structural changes in proteins freeze- or spray-dried. Monoclonal antibodies(e.g. RhuMAbVEGF) underwent secondary structural changes in the absence of a lyoprotectant. Increasing molar ratios of cryoprotectant could lead to complete structural preservation. The long-term stability of the dried proteins correlates with structural changes observed by Raman spectroscopy. 

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