Affinity purification of plasma proteins: characterization of six affinity matrices and their application for the isolation of human factor VIII.

For the purification of coagulation factor VIII, (1,1'-carbonyl-diimidazole [CDI]-activated) Sepharose CL-4B was functionalized with two aminoalkyl and four aminoalkyl-carbamylalkyl ligand-spacer combinations. The affinity matrices were contacted with human plasma. All affinity matrices showed complete adsorption of factor VIII (greater than 90%) and three aminoalkyl-carbamylalkyl Sepharoses gave factor-VIII recoveries of 50-65% and a factor-VIII preparation with a specific activity of 1-2 U factor VIII/mg of protein. Furthermore, no fibrinogen, immunoglobulin G and albumin could be detected in the isolated factor VIII. Optimal results were obtained using the di-methyl-aminopropyl-carbamyl-pentyl-Sepharose affinity matrix.

Evaluation of the physical stability of freeze-dried sucrose-containing formulations by differential scanning calorimetry.

Freeze-dried samples of sucrose with buffer salts, amino acids, or dextran have been analyzed with differential scanning calorimetry (DSC) to evaluate the use of DSC thermograms in predicting the physical storage stability. The glass transition temperature, Tg, of the amorphous cake, crystallization, and melting of sucrose are observed with DSC. Tg appeared to be an important characteristic of the physical stability of the amorphous freeze-dried cake. A storage temperature above Tg results in collapse or shrinkage of the cake, which for a sucrose-based formulation, may be accompanied by crystallization of the sucrose. The Tg of the amorphous sucrose is influenced by other components present in the cake. Dextran-40 raised Tg, while the addition of glycine to the formulation lowered Tg. The residual moisture content strongly influences Tg, since water acts as a plasticizer of the system; the higher the moisture content, the lower the Tg and the less physically stable the freeze-dried cake. Crystallization of amorphous sucrose is shown to be inhibited by high molecular weight components or ionic compounds. DSC analysis of freeze-dried cakes proved to be a powerful tool in formulation studies.

Vial lyophilization: calculations on rate limitation during primary drying.

PURPOSE: The aim of this study was to assess the rate limiting factors in the sublimation phase of freeze drying and to propose a simple model on the basis of these rate limitations. METHODS: A programmable freeze dryer was used. The load consisted of vials of varying size and various contents. To increase heat transfer, conductive paste was applied while the resistance toward mass transport was varied by using different restrictive capillaries. RESULTS: It was found that heat transfer limits the rate of sublimation. Presence of the commonly used excipient mannitol did not have a consequence on the rate of sublimation. The same applied for the restrictions towards mass transport. It was found that there exists not only a barrier against heat transport under the vial, but also between the glass wall and the frozen solution. CONCLUSIONS: From the results, a set of equations is proposed that enables to predict optimum sublimation conditions. For the pharmaceutical technologist this can serve as a simple and useful tool to derive a suitable freeze drying program.

Large-scale purification of factor VIII by affinity chromatography: optimization of process parameters.

The optimization of a new process for the extraction of human coagulation factor VIII (FVIII) from plasma with the tailor-made affinity matrix dimethylamino-propylcarbamylpentyl-Sepharose CL-4B (C3-C5 matrix) is described. First, plasma is applied to DEAE-Sephadex A-50 anion exchanger in order to separate a number of proteins, including coagulation factors II, IX and X (prothrombin complex), from FVIII. Subsequently, the unbound fraction of the ion exchanger, containing FVIII, is contacted with the C3-C5 affinity matrix. Optimization of the FVIII affinity chromatographic procedure is accomplished in terms of the ligand density of the matrix, adsorption mode (batch-wise versus column-wise adsorption and matrix to plasma ratio), and conditions of pH and conductivity to be applied on washing and desorption. In scale-up experiments, by processing 20 l of plasma, the recovery (340 U VIII:C/kg plasma) and the specific activity (s.a.) (1.2 U VIII:C/mg protein) are better than those obtained by cryoprecipitation (recovery 300 U VIII:C/kg plasma, s.a. 0.3 U VIII:C/mg protein). The newly developed process using the specially designed C3-C5 affinity matrix has potential application in the process-scale purification of FVIII.