Lyophilized protein

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. 

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]... 

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

S. J. Perstrelski, T. Arakawa, and J. F. Carpenter, Separation of freezing and drying-induced denaturation of lyophilized proteins using stress-specific stabilization. II. Structural studies using infrared spectroscopy, Arch. Biochem. Biophys., 303, 465... 

Due to the extraordinary toxicity of intact ribosome-inactivating toxins like ricin, abrin, and modeccin, purification and handling of these proteins must be done with extreme care. Even dust from crude seed powders or lyophilized proteins should be considered dangerous. During... 

Room temperature phosphorescence can be observed from dried proteins. Sheep wool keratin(47) has a phosphorescence lifetime of 1.4 s. Six lyophilized proteins were shown to exhibit phosphorescence at room temperature.(48) The spectra were diffuse, and the lifetime was non-single-exponential, which the authors interpreted as due to inhomogeneous distribution of tryptophans. As the protein was hydrated, the phosphorescence lifetime decreased. This decrease occurred over the same range of hydration where the tryptophan fluorescence becomes depolarized. Hence, these results are consistent with the idea that rigidity of the site contributes to the lifetimes. 

There are three commonly used methods [145] to incorporate enzymes in RMs (i) injection of a concentrated aqueous solution, (ii) addition of dry lyophilized protein to a reverse miceUar solution, and (iii) phase transfer between bulk aqueous and surfactant-containing organic phases. Figure 3 shows schematically the three enzyme solubiHzation methods. The injection and dry addition techniques are commonly used in biocatalytic appHcations, the latter being well suited to hydrophobic proteins [146]. The phase transfer technique is the basis for extraction of proteins from aqueous solutions. 

Protein-based drugs have been formulated mainly as stable liquids or in cases where liquid stability is limiting as lyophilized dosage forms to be reconstituted with a suitable diluent prior to injection. This is because their delivery has been limited primarily to the parenteral routes of intravenous (IV), subcutaneous (SC), or intramuscular (IM) administration. There are a few drugs that have been developed for pulmonary delivery, such as rhDNase (Pulmozyme ) and an inhalable formulation of insulin (e.g., Exubra ). However, even such drugs have been formulated as either liquid or lyophilized or spray-dried powders. This chapter will focus only on excipients that are applicable to liquid and lyophilized protein formulations. 

We begin with a brief summary of the degradation pathways of proteins, followed by a discussion on the composition of liquid and lyophilized protein formulations and on various excipients in some detail. An important feature of this chapter is a comprehensive table (Appendix), which details the formulations of approved protein drugs through the year 2005. The table has been compiled with the help of several sources (1,10,11). 

While each formulation is unique, there are several general aspects with respect to excipient components in both liquid and lyophilized protein formulations. A comparison of the excipient components in liquid and lyophilized protein formulations is provided in Table 1. 

Table 1 Excipient Components of Liquid and Lyophilized Protein Formulations... 

Carpenter JF, Chang BS, Garzon-Rodriguez W, Randolph TW. Rational design of stable lyophilized protein formulations theory and practice. Pharm Biotechnol 2002 13 109-133. 

Heparin and heparin-like polyanions are known to have significant stabilizing effects on proteins. Heparin itself has been used as a constituent of the solution used to reconstitute lyophilized proteins (54) while enoxaparin is a low molecular weight... 

Zhang MZ, et al. A new strategy for enhancing the stability of lyophilized protein the effect of the reconstitution medium on keratinocyte growth factor. Pharm Res 1995 12(10) 1447-1452. 

Lyophilize protein extracts to provide highly purified samples suitable for most subsequent experiments. 

Moy, T., Calabria, A. and Hsu, C. (2001) Near infrared A non-invasive method to determine residual moisture of lyophilized protein pharmaceuticals American Chemical Society 221st National Meeting, BIOT-016. Meeting abstract. 

Iin, T.P., Hsu, Ch.C Determination of residual moisture in lyophilized protein pharmaceuticals using a rapid and non-invasive method near-infrared spectroscopy. PDA J. Pharm. Sci. Technol. 56, pp. 196-205, 2002... 

Liu, W.R., Langer, R. Moisture induced aggregation of lyophilized proteins in the solid state. Biotechnol. Bioeng. 37, 177-184,1991... 

Davidson, P., Sun, W.Q. Effects of su-crose/raffinose mass ratios on the stability of co-lyophilized protein during storage above Tg. Pharm. Res. 18, 474-479, 2001... 

Glycogen phosphorylase b stock solution—Dissolve 25 mg ( 750 units) of glycogen phosphorylase b (Sigma catalog P-6635) in approximately 750 pi of concentrated enzyme storage buffer (resuspend the lyophilized protein gently, do not use a vortex mixer). Divide into 20 pi aliquots and store at —20°C. Dilute this 1000-unit/ml stock solution to the required concentration (see protocol) just before use. Store the dilute enzyme on ice until it is ready to be used. 

R. A. Baffi and R. L. Gamick, Quality control issues in the analysis of lyophilized proteins, Dev. Biol. Stand. 181 -184 ( 1991). 

X. MECHANISMS FOR FAILURE OF DEXTRAN TO PROTECT LYOPHILIZED PROTEINS... 

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