Lyophilized protein sequencing

Not to be overlooked when selecting components for the mobile phase are the more practical considerations such as component volatility and compatibility with enzymatic or biological assay conditions. Volatility allows easy concentration of samples using simple vacuum dryers or lyophilization. This normally means concomitantly less loss through transfer and less sample manipulation than with other concentrating systems. This type of buffer is particularly useful if the next step following purification is protein sequenc-... 

Lyophilized samples were dissolved in 20 xl 0.1% formic acid and centrifuged at 15,000 g for 5 min. Peptide sequencing of identified protein spots was performed by... 

HIV recombinant-NCp7. HIV NC coding sequence was cloned into the pMal-c vector, expressed as a fusion protein in E.coli. After factor Xa cleavage, the 55 residues NC protein (Fig. 3) was purified by HPLC and complexed with two equivalent of Zn and stored as a lyophilized powder (NCp7). 

The risk of chemical instability can be assessed from the primary sequence of the protein. The sequence containing labile amino acids such as Asn-Gly and Met would be indicative of potential instability issues. The rate of chemical reactions that alter the primary sequence of the protein is higher in solution conditions and can limit the shelf-life of protein therapeutics. As the mobility of reactants is minimized in the solid state, freeze-drying is often attempted to improve the stability [16]. In such instances, physical instability is a major issue to be dealt with. Freezedrying, also termed as lyophilization, is a dessication process in which the solvent (usually water) is first frozen and then is removed by sublimation in a vacuum [17]. In other words, the protein in solution is frozen, producing discrete ice and solute crystals. The solid ice is sublimed. Controlled heating desorbs any of the tightly bound water. 

Figure 4 Sampling plans for the determination of product uniformity within the lyophilizer. (A) Uniformity of protein content for a product in vials. (B, Q Uniformity of residual moisture for a product in ampoules. The lyophilizer has eight shelves and each shelf holds 15 trays of product containers. Arabic numerals refer to the tray number and show the order in which the trays are loaded during filling. Roman numerals relate to the sealing sequences. The trays are unloaded by blocks 12, starting from tray 120. As the trays are sealed in twos, the first two trays subject to sealing are identified by the number I, the last two trays by the number VI. The trays from which samples are collected for analysis are shaded.

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