Fractionation methods freeze-drying

Another scaffold fabrication technique is thermally induced phase separation (TIPS) [34,46,84,147-149]. TIPS involves decreasing the temperature of a polymer solution to obtain a polymer-rich and polymer-poor phase. Following phase separation, the solvent is removed using one of a number of methods (freeze drying, evaporation) resulting in the formation of pores in the polymer stracture. TIPS can also be combined with the use of porogens to increase void fraction, have better control over pore size, or improve pore interconnectivity. 

The resuspended and formulated Fraction II precipitate normally contains some aggregated IgG and trace substances that can cause hypotensive reactions in patients, such as the enzyme prekail ikrein activator (186). These features restrict this type of product to intramuscular adininistration. Further processing is required if products suitable for intravenous adininistration are required. Processes used for this purpose include treatment at pH 4 with the enzyme pepsin [9001-75-6] being added if necessary (131,184), or further purification by ion-exchange chromatography (44). These and other methods have been fiiUy reviewed (45,185,187,188). Intravenous immunoglobulin products are usually suppHed in the freeze-dried state but a product stable in the solution state is also available (189). 

The volatile fraction as defined by the various wet oxidation methods and most of the direct injection methods would be that fraction removed by acidification and purging with inert gas at room temperature. In the freeze-drying method of Gordon and Sutcliffe [29] the volatile fraction is that fraction lost by sublimation in vacuo. There have been no actual determinations of these losses, and for the most part Skopintsev s numbers were accepted as valid for all of these methods, largely because they are the only numbers available. 

We have employed two different protocols for the chemical fractionation of GSE obtained from MegaNatural-AZ based on the amounts needed for bioactivity-based assays. Batches of GSE (50 g) were extracted in acetone/water (7 3) under N2 with mechanical agitation for 12 h. The acetone was removed on a rotary evaporator and the aqueous phase was freeze-dried to yield 48 g of tannin crude extract (TCE). TCE was further fractionated following two different methods. 

Figure 2. Comparison of mutagenic activity in lyophilized and XAD-concentrated drinking water. The sampling 7000-fold concentration with either XAD-4/8 (XAD) or freeze-drying (FD) XAD-4/8 elution with acetone (neutral fraction) freeze-drying elution successively with acetone, ether, and DM SO and subsequent mutagenicity testing with strains TA98 and TA100 were as described in Materials and Methods. Each point represents the average of three plates, and 0.2 mL of concentrate corresponds to 1.4 L of water per plate.

Fractionation with organic solvents is mainly done to remove proteins, large peptides, and non-proteinaceous material such as fat. In a method developed by Harwalkar and Elliott (1971) and adopted by Lemieux et al. (1990), Puchades et al. (1990), and Visser et al. (1983), freeze-dried samples of cheese were extracted using methanol (to precipitate large peptides and proteins), chloroform (to remove fat), and water. The final extract... 

According to the operational definition, it is most important that well-defined methods and clear protocols are provided describing the isolation procedure applied. After isolation, freeze-drying is often used to stabilize the final fraction and to prevent chemical and biological reactions during storage. Critical reviews on the isolation and concentration techniques for aquatic substances are given by Aiken (1985), Leenheer (1985), and Abbt-Braun and Frimmel (2002). 

Undercooling is the driving force in freeze drying. An aqueous salt solution is introduced dropwise into an immiscible liquid (hexane or a petroleum fraction such as kerosene) cooled below 243 K. The individual droplets are frozen instantaneously and the solid particles are decanted or filtered. The frozen particles are then sublimed in a vacuum to obtain a homogeneous powder of fairly uniform particle size. Important parameters in freeze drying are the final temperature of the salt solution and the cooling rate. These can be controlled to some extent, but only on a small scale. Hence the method is not very suited for large-scale manufacture of catalysts. 

Emulsion Capacity and Stability. A 0.5 g sample of the freeze-dried protein fraction was redissolved in a minimum of 0.3 M citrate-phosphate buffer at pH 7.0 and mixed thoroughly with 50 ml of 1 M NaCl for 1 min in a Sorvall Omnimixer at 1000 rpm in a one pint Mason jar set in a water bath (20°C). Crisco oil (50 ml) was added to the jar and an emulsion formed by mixing at 500 rpm with simultaneous addition of oil at the rate of 1 ml/min until the emulsion broke. The endpoint was determined by monitoring electrical resistance with an ohmeter. As the emulsion broke a sharp increase (l KS2 to 35- 0 KSi) was noted. Emulsion capacity was expressed as the total volume of oil required to reach the inversion point per mg protein. This method is similar to that used by Carpenter and Saffle (8) for sausage emulsions. To establish emulsion stability the same procedure was used except that 100 ml of oil was added and a stable emulsion formed by blending at 1000 rpm for 1 min. A 100 ml aliquot was transferred to a graduate cylinder and allowed to stand at room temperature. Observations were made of the volume of the oil, emulsion and water phases at 30, 60, 90 and 180 min. 

Thurman and Malcolm (1981) proposed a large-scale preparative method for isolation of aquatic humic substances, in which XAD-8 resin is used to adsorb humic substances from acidified water samples. The aquatic humic substances are then back-eluted with NaOH, and fractionated into aquatic HAs (insoluble at pH 1) and aquatic FAs (soluble at pH 1). The two fractions are further purified to remove inorganic solutes and freeze-dried separately. 

The impact of formulation on virus reduction and product recovery (e.g., potency) was evaluated during terminal freeze dry/dry heat treatment studies with an unlicensed Fraction I derived product, that will be designated Protein G. As shown in Fig. 10, the optimum formulation, that achieved >4 logio PPV inactivation and 80% product recovery, was one that contained 2% albumin, no NaCl, and <0.3%o moisture by the Karl Fischer coulometric method. In contrast, freeze dry/dry heat treatment of product formulated with no albumin, 150mM NaCl and low moisture resulted in approximately 3 logio PPV reduction and 35% product recovery. Thus, minor changes in formulations such as the addition of 2% albumin may impact virus reduction and product recovery during a freeze dry/dry heat treatment. 

The volatile fraction is also defined by the measurement technique. As covered in the section on the determination of the dissolved fraction, the methods used for removing carbonate carbon must result in the loss of some fraction of the organic material present. This fraction is customarily considered as the volatile fraction, and it is different if it is lost by acidification and evaporation at 60 °C or by acidification followed by freeze-drying. In any case, the volatile fraction is not the fraction volatile at normal surface ocean temperature and pH but rather the fraction volatile at a pH low enough to permit the carbonate removal. In our laboratory, hydrocarbons, ethers, aldehydes, ketones, and some alcohols and acids can be removed by these techniques. 

---