Diafiltration ultrafiltration

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. 

Prothrombin complex is the combination of Factors E, VE, DC, and X. It is indicated for individuals with hemophdia B. In a typical production process, the supernatant resulting from the separation of cryoprecipitate is filtered to remove particles and applied to an anion-exchange column. The coagulation factors are eluted, ultrafiltered, and exposed to 60°C for 10 h for virus inactivation. This is followed by diafiltration, ultrafiltration, and sterile filtration. 

Monoclonal antibody 0.01-1 Precipitation/centrifugation, chromatography, diafiltration, ultrafiltration [4]... 

Some of the different ultrafiltration processes within the four process combinations include diafiltration as well. When diafiltration was included at a stage, the sequence, ultrafiltration - diafiltration - ultrafiltration, was used in order to obtain a high separation efficiency and membrane capacity (8). 

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. 

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. 

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. 

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. 

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... 

Removing Small Molecular Weight Contaminants. Ultrafiltration in hollow fiber membrane cartridges was the best method found to remove small molecular weight materials (75). It also served to concentrate the enzymes and change the buffer (via diafiltration) to that required for the next purification step. A 10,000 mwt... 

Ultrafiltration results in little loss of enzyme activity and can be nsed for concentration and fractionation on basis of molecular weight, for removal of salts and low molecnlar weight species, as well as for changing salt composition by diafiltration. 

After ion exchange chromatography the enzyme preparation is in a more workable volume and is then ready for further purification. Depending on the requirement for the next step, the enzyme solution can be desalted by gel filtration or ultrafiltration or the buffer can be changed by diafiltration. 

Industrially, whey proteins are prepared by ultrafiltration or diafiltration of whey (to remove lactose and salts), followed by spray drying these products, referred to as whey protein concentrates, contain 30-80% protein. 

Ultrafiltration/diafiltration of acid or rennet whey to remove varying amounts of lactose, and spray-drying to produce whey protein concentrates (30-80% protein). 

Ultrafiltration/Diafiltration (UF/DF) used as needed for product concentration (volume reduction) and buffer exchange (prepared for next step or for storage)... 

Fig. 5. Glucose concentration over time for diafiltration saccharification experiments at 15% (w/w) initial insoluble solids concentration and 20 FPU/g of cellulose enzyme loading. Vacuum filtration and ultrafiltration were applied at four different times to remove glucose (vertical dashed lines). Filtration was not applied to the control flasks. Error bars represent averages 1 SD for three repeated experiments.

---