Toyopearls

Heitz et al. (33) also described the preparation of polyvinyl acetate cross-linked with butanediol divinyl ether. The polymer is the base of the Merckogel series of size exclusion chromatography packings, and its hydrolyzed derivative, polyvinyl alcohol, is marketed as Fractogel and Toyopearls. 

SIZE EXCLUSION CHROMATOGRAPHY USING TSK-GEL COLUMNS AND TOYOPEARL RESINS... 

Toyopearl HW size exclusion chromatography resins are macroporous packings for bioprocessing chromatography. They are applicable for process-scale... 

Toyopearl HW resins overcome several disadvantages of conventional gels, which do not function well at higher flow rates or pressures, are unstable to extremes of pH, salt, and organic solvent concentrations, and can leach saccharide derivatives into the process material. For process-scale SEC, Toyopearl HW resins provide the following advantages ... 

TABLE 4.20 Properties and Molecular Weight Separation Ranges for Toyopearl HW Resins... 

Toyopearl resin Particle size ( tm) Pore size (A) Polyethylene glycols and oxides Dextrans Globular proteins... 

FIGURE 4.43 Calibration curves for globular proteins on toyopearl resins. Column 22 mm X 30 cm. Sample Protein standards. Elution 0.06 A1 phosphate buffer, pH 7, in 0.06 A1 KCI. Legend elution volume V column volume. 

Like other GFC matrices, including TSK-GEL SW and TSK-GEL PW packings, and dextran and agarose gels, Toyopearl HW resins exhibit some ionic and hydrophobic interaction with samples. The hydrophobic properties of Toyopearl HW resins, however, can be utilized more effectively for improved protein purifications because, unlike numerous other GFC packing materials, Toyopearl HW resins can be used with high levels of organic solvent (38). 

Purifications are made simpler with Toyopearl HW media because there is no risk of leached polysaccharides to contaminating eluted fractions. Saccharide derivatives have been known to leach from conventional low-pressure column packings, such as dextran or agarose gels. 

For many proteins, especially glycoproteins, the physical characteristics, particularly the hydrophilic nature of Toyopearl HW resins, improve mass and activity recovery rates. Toyopearl HW media do not adsorb proteins, as conventional gels can, and thus do not interfere with sample recovery (39). 

The hydrophilic surface characteristics and the chemical nature of the polymer backbone in Toyopearl HW resins are the same as for packings in TSK-GEL PW HPLC columns. Consequently, Toyopearl HW packings are ideal scaleup resins for analytical separation methods developed with TSK-GEL HPLC columns. Eigure 4.44 shows a protein mixture first analyzed on TSK-GEL G3000 SWxl and TSK-GEL G3000 PWxl columns, then purified with the same mobile-phase conditions in a preparative Toyopearl HW-55 column. The elution profile and resolution remained similar from the analytical separation on the TSK-GEL G3000 PWxl column to the process-scale Toyopearl column. Scaleup from TSK-GEL PW columns can be direct and more predictable with Toyopearl HW resins. 

The methacrylic backbone structure makes the spherical Toyopearl particles rigid, which in turn allows linear pressure flow curves up to nearly 120 psi (<10 bar), as seen in Fig. 4.45. Toyopearl HW resins are highly resistant to chemical and microbial attack and are stable over a wide pH range (pH 2-12 for operation, and from pH 1 to 13 for routine cleaning and sanitization). Toyopearl HW resins are compatible with solvents such as methanol, ethanol, acetone, isopropanol, -propanol, and chloroform. Toyopearl HW media have been used with harsh denaturants such as guanidine chloride, sodium dodecyl sulfate, and urea with no loss of efficiency or resolution (40). Studies in which Toyopearl HW media were exposed to 50% trifluoroacetic acid at 40°C for 4 weeks revealed no change in the retention of various proteins. Similarly, the repeated exposure of Toyopearl HW-55S to 0.1 N NaOH did not change retention times or efficiencies for marker compounds (41). 

With soft gels, column packing has often been plagued with such problems as inferior reproducibility and excessive time requirements. These problems are alleviated with physically stable Toyopearl HW media. However, an improperly packed column can have significantly reduced efficiency. The two key variables for the successful packing of Toyopearl HW media, packing velocity and column size, have been evaluated to determine the optimal packing conditions. 

Figure 4.46 shows the relationship between packing velocity and sample resolution using Toyopearl HW-55F and Toyopearl HW-55S columns and... 

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