Diafiltration

Ultrafiltration may also be utilized to achieve a number of other objectives. As discussed above, it may yield a limited degree of protein purification and may also be effective in depyrogenating solutions. This will be discussed further in Chapter 7. The technique is also widely used to remove low molecular mass molecules from protein solutions by diafiltration. 

Diafiltration is a process whereby an ultrafiltration system is utilized to reduce or eliminate low molecular mass molecules from a solution and is sometimes employed as part of biopharmaceuti-cal downstream processing. In practice, this normally entails the removal of, for example, salts, ethanol and other solvents, buffer components, amino acids, peptides, added protein stabilizers or other molecules from a protein solution. Diafiltration is generally preceded by an ultrafiltration step to reduce process volumes initially. The actual diafiltration process is identical to that of ultrafiltration, except for the fact that the level of reservoir is maintained at a constant volume. This is achieved by the continual addition of solvent lacking the low molecular mass molecules that are to be removed. By recycling the concentrated material and adding sufficient fresh solvent to the system such that five times the original volume has emerged from the system as permeate, over 99 

If the crossflow filtration stem is well mixed, the concentration of solute in the system and the permeate Cp are equal at any instant in time and are given by a stirred tank model  

As can be seen from fig. VIH - 31. after a pre-concentration step the retentate is diluted with solvent until the desired purification has been obtained. 

The total volume of water at time t is given by and substimtion of eq. VUI - 32 into eq.vni - 31 gives 

As the membrane is fieely permeable to low molecular weight solutes (R = 0), then eq. vm - 33 indicates that 37% of the low molecular solute is still present with an amount of water equal to the initial volume and that at least five times the initial volume is needed to remove more than 99% of the low molecular weight solute (or to reduce die ratio c//Cr° to less than 0.01). S ince the membrane has a certain retention coefficient for the low molecular component, even more water is needed than predicted above. In practice, the membrane does not exhibit complete retention for one component whilst being freely permeable to the other. 

vm - 33 is very similar to that derived for a CSTR. Indeed, by setting R = 0 (no membrane ), eq. Vm - 33 reduces to the CSTR equation  

However, no fractionation is obtained with a CSTR because both high and low molecular weight solutes are washed out. 

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Membrane-retained components are collectively called concentrate or retentate. Materials permeating the membrane are called filtrate, ultrafiltrate, or permeate. It is the objective of ultrafiltration to recover or concentrate particular species in the retentate (eg, latex concentration, pigment recovery, protein recovery from cheese and casein wheys, and concentration of proteins for biopharmaceuticals) or to produce a purified permeate (eg, sewage treatment, production of sterile water or antibiotics, etc). Diafiltration is a specific ultrafiltration process in which the retentate is further purified or the permeable sohds are extracted further by the addition of water or, in the case of proteins, buffer to the retentate. 

Diafiltration is an ultrafiltration process where water or an aqueous buffer is added to the concentrate and permeate is removed (50). The two steps may be sequential or simultaneous. Diafiltration improves the degree of separation between retained and permeable species. 

Constant-volume batch diafiltration is the most efficient process mode. Eor species that freely permeate the membrane. 

Area—time requirements for a specific diafiltration mission are defined as... 

The optimum concentration for any diafiltration (minimum area time) is the minimum of the plot ... 

Sequential batch diafiltration is a series of dilution—concentration steps. The concentration of membrane-permeable species is... 

Composition and Methods of Manufacture. The vaccine consists of a mixture of purified capsular polysaccharides from 23 pneumococcal types that are responsible for over 90% of the serious pneumococcal disease in the world (47,48). Each of the polysaccharide types is produced separately and treated to remove impurities. The latter is commonly achieved by alcohol fractionation, centrifugation, treatment with cationic detergents, proteolytic en2ymes, nucleases or activated charcoal, diafiltration, and lyophili2ation (49,50). The vaccine contains 25 micrograms of each of the types of polysaccharide and a preservative such as phenol or thimerosal. 

Fig. 1. Schematic of a process for batch suspension culture of mammalian cells, where UF is ultrafiltration and DF is diafiltration.

Until the early 1960s, laboratory iavestigators rehed on dialysis for the separation, concentration, and purification of a wide variety of biologic fluids. Examples iaclude removal of a buffer from a proteia solution or concentrating a polypeptide with hyperosmotic dialysate. Speciali2ed fixtures were sometimes employed alternatively, dialysis tubes, ie, cylinders of membrane about the si2e of a test tube and sealed at both ends, were simply suspended ia a dialysate bath. In recent years, dialysis as a laboratory operation has been replaced largely by ultrafiltration and diafiltration. 

Ultrafiltration (qv) (uf) is increasingly used to remove water, salts, and other low molecular-weight impurities (21) water may be added to wash out impurities, ie, diafiltration. Ultrafiltration is rarely used to fractionate the proteins because the capacity and yield are too low when significant protein separation is achieved. Various vacuum evaporators are used to remove water to 20—40% dry matter. Spray drying is used if a powdery intermediate product is desired. Tyophilization (freeze-drying) is only used for heat-sensitive and highly priced enzymes. 

The simplest ultrafiltration is the stirred cell, a batch operation. The most compex is a continuous stages-in-series operation incorporating diafiltration. Industrial practice incorporates the full gamut of complexity. 

Diafiltration If a batch process is run so that the permeate is replaced by an equal volume of fresh solvent, unretained solutes are flushed through the system more efficiently. A major use of UF is fractionation, where a solvent, a retained solute and an unretained solute are present. An example is whey, containing water, protein, and lactose. If the retention of protein is I and the retention of lactose is 0, the concentration of protein in the retentate rises during UF. The ratio of protein to lac tose rises, but the feed concentration of lactose is unchanged in retentate and permeate. Diafiltration dilutes the feed, and permits the concentration of lactose to be reduced. Diafiltration is used to produce high-purity products, and is used to fractionate high-value products. R is always 0 for eveiy component. 

The combination of diafiltration and batch concentration can be used to fractionate two macrosolutes whose retentions differ by as little as 0.2. It is possible in principle to achieve separations that are competitive with chromatography. When tanks and other equipment are considered, as well as the floor space they occupy, the economics of membrane separation of proteins may be attractive [R. van Reis, U.S. Patent 5,256,294 (1993)]. 

Membrane Processes Membrane processes are also used diafiltration is convenient for the removal of small contaminating species such as salts and smaller proteins, and can be combined with subsequent steps to concentrate the protein. Provided that proper membrane materials have been selected to avoid protein-membrane interactions, diafiltration using ultrafiltration membranes is typically straightforward, high-yielding and capital-sparing. These operations can often tolerate the concentration or the desired protein to its solu-bihty limit, maximizing process efficiency. 

Sweeney, S.F., Woehrle, G.H. and Hutchison, J.E. (2006) Rapid purification and size separation of gold nanoparticles via diafiltration. Journal of the American Chemical Society, 128, 3190-3197. 

DiajUtration After concentration has removed permeable components from the system, a new buffer can be added to dilute the retained product back to its original volume. Repeated application of this procedure, known as batch diafiltration, is used to exchange one buffer for another and is conveniently implemented at lab scale. 

System sizing involves integration of Eq. (20-69) using a flux model to give Eq. (20-71), where Vp is the permeate volume and J is the average flux. Note the direct tradeoff between area and process time. Table 20-19 shows the concentration and diafiltration steps separated and processing time. 

The formulas for concentration and diafiltration can be combined for the entire process to derive an expression for the loss of product in the permeate. This loss is shown in Fig. 20-58 and Table 20-19 for... 

Retentate product recovery employs the batch, fed-batch, or continuous processing systems, and concentration or diafiltration modes of operation (see general membrane section). Beer and wine dealcoholization uses batch diafiltration. 

Batch or Fed-Batch Operation This mode of operation is typical of biologicals and juice processing where high solicfs and low fluxes require multiple passes, and batch operation is characteristic of the manufacturing process. Formulas in Table 20-19 can be used to calculate the required volume reduction factor X, diafiltration volumes... 

The effectiveness of the elution step can be tailored by using a single eluent, pulses of different eluents, or eluent gradients. These systems are generally characterized by mild desorption conditions. If the eluting agent is bound to the protein, it can be dissociated by desalting on a gel filtration column or by diafiltration. 

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