TABLE 410 Estimates of cell diameters and relative density with respect to medium

Relative Density (between Cell and Cell Type Diameter (mm) Medium, kg/m3)

Bacterium 1-2 70

Yeast 7-10 90

Mammalian 40-50 70

Fermentation or mass cell-cultivation

Cell suspended in medium harvested

Cell suspended in medium harvested

Cell disruption
Figure 4.13. Schematic presentation of process stream to purify a recombinant protein, starting from cell suspension harvested from fermenter or cell-cultivation vessels.

cedures also apply to the isolation and characterization of monoclonal antibodies produced by hybridoma cells.

The downstream process, starting with the biomass--cells and medium har vested from the fermentation and mass-cultivation process—can be roughly divided into four stages (Figure 4.13) [4,5]. They are (1) solid-liquid separation or clarification, (2) concentration, (3) purification, and (4) quality assurance and control analyses. These downstream processes and quality control procedures are essential to ensure that the purified recombinant product produced in a given batch meet or exceed the purity and quality established for a given pharmaceutical product.

Solid-Liquid Separation

Separation from culture media or broth is the primary step in collecting the product found either in cells (solid) or medium (liquid). This initial separation step is engineered based on cell size and density differences between solid and liquid (Table 4.10). In the case where the recombinant product is localized in the intracellular content such as the cytoplasm or inclusion bodies, which are highly insoluble particles found in bacteria, the cells are first isolated from the medium and then disrupted to collect the recombinant protein fraction. A number of cell disruption techniques have been developed to facilitate this step, and some are listed in Table 4.11.

Based on the differences between the solid and liquid density, batch centrifu-gation techniques are often used in the discovery phase as a convenient tool to separate the cell from the medium. Highspeed ultracentrifugation instruments have

■TABLE 4.11. Some methods designed to disrupt cells

■TABLE 4.11. Some methods designed to disrupt cells

Mechanical Methods

Other Methods

Ultrasonic

Drying

Homogenization

Heat or osmotic shock

Agitation with glass

Freeze-thaw

beads or abrasive

Organic solvent

materials

Chaotropic agents

Enzymes

Surfactant

been developed to separate cell membrane from the medium, even when density differences are small. However, this strategy, while used in industrial scale preparations, is less attractive because of low capacity and the high cost of maintenance and energy for operating these instruments. Engineering improvements that permit centrifugation in a continuous mode include (1) the tubular bowl, (2) multi-chamber bowl, (3) disk stack bowl, and (4) decanter or scroll centrifuge (Figure 4.14). These improvements have overcome some of the capacity limitations of centrifuga-tion, but the cost of the operation remains high.

Alternatively, separation of cells from media can be achieved with the filtration of cell suspension through membranes with defined pore size [6]. This approach takes advantage of the particle size based on size differences between cells (2-10 mm in diameter) and media (colloids of less than a few nm in diameter). Many types of filtration designs and membrane supports are available, as well as a wide range of pore sizes, to aid large-scale filtration (Figure 4.15).

Filters have been designed to reduce the fouling or clogging of solid materials at the surface of the membrane by taking into account fluid flow. Among them are the rotary drum vacuum filter (designed for collecting yeast cells in high capacity), and the cross-flow or tangential flow filter. They have been used successfully to separate solid from liquid for harvesting fermentation products. Additional design improvements in the fabrication of membrane supports, including the hollow-fiber system, and the precision of pore size have allowed implementation of ultrafiltration strategies to separate fine particles such as membrane debris and inclusion bodies (1-5 nm in diameter) from soluble materials (less than 0.5 nm).

Other strategies for separation of solid and liquid include flocculation, a method to increase sold particle size by allowing the formation of complexes with polycations, such as cationic cellulose and polymers, inorganic salts, or mineral hydrocolloids. The resulting aggregated cells or agglomerates can be separated by gravitational sedimentation, low-speed centrifugation, or filtration with much less effort. It is interesting to note that some of these cationic agents also clear pyrogens, nucleic acids, and acidic proteins that may be problematic in purification of proteins using column chromatography (Figure 4.15).

Concentration of Putative Product before Purification

After removal of cells and debris from the culture or medium though solid-liquid separation, the fraction of recombinant protein in the liquid phase usually ranges from 2% to 15%. The large volumes associated with these low concentrations make it impractical to proceed to the next step in purification. Indeed, the volume may far exceed the capacity for chromatographic purification techniques used to isolate recombinant proteins from other soluble cellular contaminants. Therefore, a concentration step is used to reduce the volume and thereby increase the recombinant

Liquid discharge

Supernantant

Soldid

Cell lysate feed inlet

Liquid discharge

Liquid discharge

Liquid discharge

Solid discharge (b)

Cell lysate feed inlet

Liquid discharge

Solid discharge (b)

Cell lysate feed inlet

Liquid discharge (higher density)

Liquid discharge (higher density)

Liquid discharge (light density)

Disk Stack Distributor

Liquid discharge (light density)

Disk Stack Distributor

Solid discharge

Cell lysate feed inlet

Liquid Solid discharge discharge

A. Tubular bowl configuration is the simplest sedimentation centrifuge and is often used in pilot pants. Due to the slender shape and small volume, a relatively high centrifugal force (g value) can be achieved with such centrifuges. The tubular bowl can be modified to continuously discharge the solid collected at the bottom of the bowl. The liquid can be collected efficiently through the top liquid discharge nozzle.

B. Multi-chamber centrifuge is a modification of the tubular bowl configuration. It contains a number of concentric screens that allow collection of particulate matter in the cell lysate and discharge from the instrument by gravitational force. The centrifugal force slings liquid through the effluent liquid discharge outlets.

C. Disc stack centrifuge contains a set of conical discs separated by flow channels, dividing the bowl into separate settling zones. Under the centrifugal force, the particles in the cell lysate (fed through inlet) are thrown into the space outside the disc stack at higher efficiency than the multi-chamber bowl configuration. The clarified liquid moves inward and upward to reach the annular discharge outlets where liquid of higher and lower densities can be collected. The solid accumulated at the bottom, outside of the disc stack, can be configured to discharge continuously through a nozzle or intermittently through a peripheral or axial eject mechanism.

D. Decanter or scroll centrifuge can be used for cell lysates with high solid content that can be as high as 80% of the suspension. Solids matters in the cell lysate are continuously compressed due to the rotation of the bowl and the closely fitted, tapered helical screw, operating at slightly different speed. The concentrated solids are discharged as they move toward the narrow end of the horizontal bowl.

Figure 4.14. Separation of cell lysates containing pharmaceutical products, employing large-capacity centrifuges that are configured with tubular bowl (A), multi-chamber bowl (B), disc stack bowl (C), or decanter (scroll) bowl (D).

Figure 4.15. Solid-liquid separation in industrial scale using centrifugation in continuous mode (A) or passage of the suspension through manifolds such as those shown in (B) mounted with filter cartridges (C) designed for tangential or cross-flow of liquid suspension. Panel A: NIH Fredrick facility, with permission; Panels B and C: Milipore, MA, with permission.

Figure 4.15. Solid-liquid separation in industrial scale using centrifugation in continuous mode (A) or passage of the suspension through manifolds such as those shown in (B) mounted with filter cartridges (C) designed for tangential or cross-flow of liquid suspension. Panel A: NIH Fredrick facility, with permission; Panels B and C: Milipore, MA, with permission.

protein concentration to make it suitable for chromatographic conditions.

Heat-assisted evaporation strategies, such as the falling-film evaporator, plate evaporator, forced-film evaporator, and centrifugal forced-film evaporator have been developed and are used to remove water from solutions of small peptides, such as antibiotics. But most recombinant proteins are heat labile and may not survive this strategy.

Other strategies have been developed to reduce the volume of recombinant protein solutions [7]. Some of the agents and strategies used to accomplish this are listed in Table 4.12 [7,8]. One approach is to precipitate the protein from solution, followed by reconstitution into a small volume of solvent.

Protein precipitation, using agents that decrease the solubility of the recombinant product, is an established technique in the pharmaceutical industry. While salts and organic solvents have traditionally been used, more specific precipitation reagents are now being tested to improve protein purification. These specific reagents are designed to provide noncovalent cross-linking of proteins, resulting in protein aggregates that precipitate from solution. They include antibodies for affinity precipitation and homo- and heterobifunc-tional ligands that bind to the recombinant protein, and other carriers [9]. Regardless

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