Cell conventional filtration

Pharmaceutical Removal of suspended matter is a frequent application for MF. Processes may be either clarification, in which the main product is a clarified liquid, or solids recovery. Separating cells or their fragments from broth is the most common application. Clarification of the broth in preparation for product recovery is the usual objective, but the primary goal may be recovery of cells. Cross-flow microfiltration competes w l with centrifugation, conventional filtration by rotary vacuum filter or filter press and decantation. MF delivers a cleaner permeate, an uncontaminated, concentrated cell product... 

Microfiltration can be categorized between conventional filtration and UF. The process is used to filter very small particles (usually <10 pm in size) from a suspension, by using a membrane with very fine pores. Example of microfiltration include the separation of some microorganisms from their suspension, and the separation of blood cells from whole blood, using a microporous membrane. 

In conventional filtration systems used for cell separation, plate filters (e.g., a filter press) and/or rotary drum filters are normally used (cf. Chapter 9). The filtrate fluxes in these filters decrease with time due to an increase in the resistance of the cake Rc (m 1), as shown by Equation 9.1. If the cake on the filtering medium is incompressible, then Rc can be calculated using Equation 9.2, with the value of the specific cake resistance a (m kg-1) given by the Kozeny-Carman equation (Equation 9.3). For many microorganisms, however, the values of a obtained by dead-end filtration (cf. Section 9.3) are larger than those calculated by Equation 9.3, as shown... 

In conventional population cell-based assays, sample preparation involves several steps, including lysis, filtration of non-solubUized cellular material, labeling of the analytes of interest, and sample purification. However, to prevent dilution of the extremely small samples in single-cell analysis, filtration and purification are not typically integrated in chemical cytometiy methods. This section briefly describes the various cell lysis techniques which have been implemented in microfluidics for more detail, please see On-Chip Cell Lysis. 

Favre (1993) found that his data for constant volume perfusion of two different mouse-mouse hybridoma lines could not fit the classical constant volume filtration equation. He suggested that this could be due to a monolayer type of cell buildup instead of a cake in the conventional filtration. According to Favre, a model principally based on stochastic and steady plugging of the pores in the screen by the cells leading to an equilibrium state can possibly give a rational design procedure for spin filters. 

Conventional filtration is used to separate large volumes of diluted cells suspensions (>1 L), extracellular products and mould suspensions because the mycelium has very low density and it is difficult to separate the fluid by centrifugation. ... 

Example 12.1 A batch conventional filtration equipment is used to filter a cell culture suspension which has a viscosity of 3.0 cp. The data for fitration (Table 12.1) at constant pressure is obtained with a vacuum pressure of 500 mm HG. The cell solids on the filter at the end of filtration were dried and found to weigh 14 grams. Determine the specific cake resistance, r and the medium resistance Rm- Estimate how long it would take to obtain 5000 liters of filtrate from this ceU broth on a filter with a surface area of 12 and vacuum pressure of 450 mm Hg. The final volume of the cake is found to be 690 ml. 

No internal piping and no conventional filter valve are needed with single-cell dmm filters where the entire dmm also operates under vacuum. The cake discharge is effected by air blowback from an internal stationary shoe mounted inside the dmm at the point of discharge. There are very close tolerances between the inside surface of the dmm and the shoe in order to minimize the leakage. The inside of the dmm acts as a receiver for the separation of air and filtrate conventional multicompartment dmm filters require a separate external receiver. This type of filter permits operation of the filter with thin cakes so that high dmm speeds, up to 26 rpm, can be used and high capacities can be achieved. Sizes up to 14 m are available. 

PVDF-based microporous filters are in use at wineries, dairies, and electrocoating plants, as well as in water purification, biochemistry, and medical devices. Recently developed nanoselective filtration using PVDF membranes is 10 times more effective than conventional ultrafiltration (UF) for removing vimses from protein products of human or animal cell fermentations (218). PVDF protein-sequencing membranes are suitable for electroblotting procedures in protein research, or for analyzing the phosphoamino content in proteins under acidic and basic conditions or in solvents (219). 

It is advantageous to generate bubbles of micron-size when the particles to be floated are very small. The generation of such bubbles is almost impossible in conventional equipment which relies on mechanical means of breaking down the gas. If air, or another gas, is dissolved under pressure in the suspension before it is introduced into the cell, numerous microbubbles are formed when the pressure is reduced and these then attach themselves to the hydrophobic particles. Similar effects can be obtained by operating the cells under vacuum, or producing gas bubbles electrolytically. Dissolved and electro filtration are discussed later. 

Kempken et al. [113] employed a rotating disc filter to harvest CHO cells, and observed that the filter could be operated at low transmembrane-pressure with high wall shear rates, leading to high filtrate flow rates, high product yields and minimum fouling. They concluded that their system offered a powerful alternative to conventional tangential flow filtration. 

The purification of bacterial constituents usually starts in a very conventional way with an extraction step of the crude broth at neutral or slightly acidic pH. Mycelium-forming organisms are separated by filtration, and the cell mass and the filtrate are extracted separately. For the liquid phase, adsorber resins allow high recovery rates of metabolites and low process costs due to repeated use of the resins. If liquid-liquid extraction has to be applied, medium or highly polar solvents are favored. Ethyl acetate is the solvent of choice, and only in few cases is butanol superior. To extract the moist cell material, ethyl acetate, acetone or dichloromethane/methanol can be used. 

The filtrate is further dehydrated in 100% hexamethyldisilazine (HMDS) for 20 sec at 37°C in a microwave oven at the same power level utilizing the temperature probe in the blank Petri dish containing HMDS. The filtrate is dried in a conventional oven for 15 min at 60°C. After 15 min all excess HMDS is removed, and the filter with attached cells is allowed... 

Arce et al. [39] developed a flow injection analysis (F1A) system (Fig. 5.3) for online filtration of water samples prior to CE analysis. They also constructed a pump-driven unit for extraction and filtration of soil samples combined with CE in an online mode (automated sample transfer between pre-CE sample preparation step and the CE) [40]. The method was precise and four times faster than conventional methods of sample preparation with an off-line unit. Blood samples are centrifuged immediately to remove red blood cells and the serum is stored as discussed above. Sometimes, urine samples also contain precipitates which are removed by centrifuge. 

ECB deacylase is produced naturally by Actinoplanes utahensis. However, very little (—0.2%) deacylase activity was detected in the culture filtrate of Actinoplanes utahensis. Less than 5% of the cell-associated deacylase activity was released by incubation of the cells at 0.01 M KH2P04, pH 6, for 1 day. A simple salt treatment with 0.8 M KC1 resulted in 60-80% recovery of soluble deacylase at a high specific activity [25], This salt-induced solubilization suggests that the deacylase is loosely bound to the membrane of A. utahensis and can be released by disruption of ionic interactions [26]. The solubilized enzyme was stable and purified to apparent homogeneity by a four-step conventional procedure [25]. 

Conventional pressure or vacuum filtration techniques are widespread in industry for separating cells and other biological materials from a liquid phase which can be solvent based or aqueous. A pressure differential between the dirty and clean sides of the filter, created with over pressure or vacuum, provides a driving force for the liquid to be forced through the filter material which retains solids above a particular size. This type of filter is often used in conjunction with a precoat material on the filter to improve the separation characteristics. 

---