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Kieselguhr-agarose composite medium for downstream absorption
TIBTECH - OCTOBER 1986
In a study in which a 1.6 x 30cm column of Macrosorb DEAE was used for the isolation of BSA from bovine serum, increasing the linear flow rate from 1 cm/min to 5 cm/min gave a separation showing no loss of resolution. There was Kieselguhr-agarose composite medium for downstream no decrease in the yield and purity of the BSA isolated throughout twelve conadsorption tinuous cyclic runs. Linear flow rates of Newly available composite adsorbents simpler equipment at all scales. Gener- 10 cm/min produced significant process (Macrosorb) circumvent some problems ally, the flow rates used with traditional time savings without great buffer overwhich arise when compressible soft gel soft gels are limited not by the kinetics of usage. media are used as column packings. The the adsorption/desorption processes, but The silica 'backbone' of Macrosorb also materials consist of rigid kieselguhr- by the poor mechanical properties increases the density of the composites, based granules which contain an inter- characteristic of such media. The kiesel- allowing them to be used in fluidised connected macroporous interior (radius guhr (silica) skeleton of the granules has a beds, both in the presence and absence of 7 ~tm) filled with electrophoretic grade low surface activity and exhibits nnde- cell debris. Fluidised bed configurations agarose. The composites form stable beds tectable non-specific adsorption of pro- permit processing of large stock volumes which do not change volume on change of teins. at rates which would not normally be solvent. Derivatisation of the agarose Separations from non-ideal mixtures considered, and enable dilution to yields ion-exchangers and affinity media. using these composite ion-exchangers replace dialysis as a method of reducing Macrosorb ion-exchangers have been demonstrate that the purity of the agarose salt concentration before adsorption. shown to have characteristics similar to used reduces non-specific adsorption. those of traditional soft agarose media, Consequently, purer products are ob- For further information contact Dr F. J. S. but with a number of physical advant- tained, and if the adsorbent is used Reed at Sterling Organics, Dudley, Cramages. repetitively, increased throughput results lington, Northumberland NE23 7QG, Dramatically improved flow rates at from the reduced demand for scrubbing England. Tel: (091) 250 0471. low pressures allow faster isolations in and associated re-equilibrations.
Screening method for antitumour agents A number of natural products which maintain the integrity of the spindle inhibit the synthesis and functioning of proteins. The slides were examined microthe mitotic spindle have been shown to be potent antitumour agents. Many of these scopically for a number ' o f features agents act on the dimers of tubulin including spindle shortening or absence, proteins of the spindle during cell abnormal chromosome divisions, altered division. anaphase-telophase/metaphase ratio and A screening system was developed increase in multipolar spindles. It was found that the screening system using a primary line of fibroblast cells, HSBP. Suspensions of cultured cells were was extremely sensitive for detection of allowed to attach to the surface of sterile spindle toxins from complex plant slides contained in petri dishes. During extracts (down to 2 ~tm/ml). the active growth phase, the agent to be tested was added and the cells incubated for a further 28 h. Cells were then stained Somers A., Parry, J.M., Parry, E.M., differentially (spindles blue, chromo- Stafford, A. and Kelly, S.L. (1986) somes red) under conditions designed to J. Biotochnol. 4, 219-233
Process control measures enable the culture of melanoma cells free from macromolecular growth factors The use of serum in large scale culture of mammalian cells presents problems of serum supply, foaming, downstream processing and cost, Serumfree media usually require supplementation with expensive growth-promoting macromolecules. Certain cells, however, can grow without serum or macromolecular growth factors but, on a large scale, conditions must be carefully controlled. The melanoma cell line Bowes 4 was cultured for more than five months in serum-free/macromolecule-free medium
using a mechanically stirred loop bioreactor mixed with marine impellers; pO2 was controlled at 125 mbar and the pH at 7.15. Cell number could be correlated with the signal of an in-line fluorimeter, and CO2 and redox potential were monitored by on-line sensors. Both the growth rate and cell number of the culture under these conditions were close to those found in medium with serum. Leist, C., Meyer, H-P. and Fiechter, A. (1986) J. Biotechnol. 4,235-246
~) 1986, Elsevier Science Publishers B.V., Amsterdam 0166-9430/86/$02.00
Enhanced nitrogen fixation and nodulation in Rhizobium loft Rhizobium spp. contain genes coding for symbiotic functions-(root nodule development and N2 fixation) in legumes. In R. meliloti and R. leguminosarum, these genes are located on large indigenous plasmids. R. loti also contains a large indigenous plasmid but (in the two strains so far examined) the plasmid does not carry the symbiotic genes: Nz-fixing root nodules were still formed on Lotus spp. by a R. loti strain cured of its plasmids. The plasmid-cured strain (PN4010) was obtained by heat treatment; another R. ]oti strain (PN4012) was created by restoring a derivative of the lost plasmid into PN4010. It could be shown that on certain strains of Lotus plants, the plasmid-cured strain of R. loti showed higher nitrogen fixation effectiveness than either of the plasmid-containing strains (strain NZP2037 from which PN4010 was derived, and PN4012). The increased effectiveness appeared to be associated with an increased production of nodules. The plasmid-cured strain was also a more efficient competitor for nodulation than the plasmid-containing strains. Pankhurst, C. E., McDonald, P. E. and Reeves, J.M. (1986) J. Gen. MicrobioL 132, 2321-2328
In a study in which a 1.6 x 30cm column of Macrosorb DEAE was used for the isolation of BSA from bovine serum, increasing the linear flow rate from 1 cm/min to 5 cm/min gave a separation showing no loss of resolution. There was Kieselguhr-agarose composite medium for downstream no decrease in the yield and purity of the BSA isolated throughout twelve conadsorption tinuous cyclic runs. Linear flow rates of Newly available composite adsorbents simpler equipment at all scales. Gener- 10 cm/min produced significant process (Macrosorb) circumvent some problems ally, the flow rates used with traditional time savings without great buffer overwhich arise when compressible soft gel soft gels are limited not by the kinetics of usage. media are used as column packings. The the adsorption/desorption processes, but The silica 'backbone' of Macrosorb also materials consist of rigid kieselguhr- by the poor mechanical properties increases the density of the composites, based granules which contain an inter- characteristic of such media. The kiesel- allowing them to be used in fluidised connected macroporous interior (radius guhr (silica) skeleton of the granules has a beds, both in the presence and absence of 7 ~tm) filled with electrophoretic grade low surface activity and exhibits nnde- cell debris. Fluidised bed configurations agarose. The composites form stable beds tectable non-specific adsorption of pro- permit processing of large stock volumes which do not change volume on change of teins. at rates which would not normally be solvent. Derivatisation of the agarose Separations from non-ideal mixtures considered, and enable dilution to yields ion-exchangers and affinity media. using these composite ion-exchangers replace dialysis as a method of reducing Macrosorb ion-exchangers have been demonstrate that the purity of the agarose salt concentration before adsorption. shown to have characteristics similar to used reduces non-specific adsorption. those of traditional soft agarose media, Consequently, purer products are ob- For further information contact Dr F. J. S. but with a number of physical advant- tained, and if the adsorbent is used Reed at Sterling Organics, Dudley, Cramages. repetitively, increased throughput results lington, Northumberland NE23 7QG, Dramatically improved flow rates at from the reduced demand for scrubbing England. Tel: (091) 250 0471. low pressures allow faster isolations in and associated re-equilibrations.
Screening method for antitumour agents A number of natural products which maintain the integrity of the spindle inhibit the synthesis and functioning of proteins. The slides were examined microthe mitotic spindle have been shown to be potent antitumour agents. Many of these scopically for a number ' o f features agents act on the dimers of tubulin including spindle shortening or absence, proteins of the spindle during cell abnormal chromosome divisions, altered division. anaphase-telophase/metaphase ratio and A screening system was developed increase in multipolar spindles. It was found that the screening system using a primary line of fibroblast cells, HSBP. Suspensions of cultured cells were was extremely sensitive for detection of allowed to attach to the surface of sterile spindle toxins from complex plant slides contained in petri dishes. During extracts (down to 2 ~tm/ml). the active growth phase, the agent to be tested was added and the cells incubated for a further 28 h. Cells were then stained Somers A., Parry, J.M., Parry, E.M., differentially (spindles blue, chromo- Stafford, A. and Kelly, S.L. (1986) somes red) under conditions designed to J. Biotochnol. 4, 219-233
Process control measures enable the culture of melanoma cells free from macromolecular growth factors The use of serum in large scale culture of mammalian cells presents problems of serum supply, foaming, downstream processing and cost, Serumfree media usually require supplementation with expensive growth-promoting macromolecules. Certain cells, however, can grow without serum or macromolecular growth factors but, on a large scale, conditions must be carefully controlled. The melanoma cell line Bowes 4 was cultured for more than five months in serum-free/macromolecule-free medium
using a mechanically stirred loop bioreactor mixed with marine impellers; pO2 was controlled at 125 mbar and the pH at 7.15. Cell number could be correlated with the signal of an in-line fluorimeter, and CO2 and redox potential were monitored by on-line sensors. Both the growth rate and cell number of the culture under these conditions were close to those found in medium with serum. Leist, C., Meyer, H-P. and Fiechter, A. (1986) J. Biotechnol. 4,235-246
~) 1986, Elsevier Science Publishers B.V., Amsterdam 0166-9430/86/$02.00
Enhanced nitrogen fixation and nodulation in Rhizobium loft Rhizobium spp. contain genes coding for symbiotic functions-(root nodule development and N2 fixation) in legumes. In R. meliloti and R. leguminosarum, these genes are located on large indigenous plasmids. R. loti also contains a large indigenous plasmid but (in the two strains so far examined) the plasmid does not carry the symbiotic genes: Nz-fixing root nodules were still formed on Lotus spp. by a R. loti strain cured of its plasmids. The plasmid-cured strain (PN4010) was obtained by heat treatment; another R. ]oti strain (PN4012) was created by restoring a derivative of the lost plasmid into PN4010. It could be shown that on certain strains of Lotus plants, the plasmid-cured strain of R. loti showed higher nitrogen fixation effectiveness than either of the plasmid-containing strains (strain NZP2037 from which PN4010 was derived, and PN4012). The increased effectiveness appeared to be associated with an increased production of nodules. The plasmid-cured strain was also a more efficient competitor for nodulation than the plasmid-containing strains. Pankhurst, C. E., McDonald, P. E. and Reeves, J.M. (1986) J. Gen. MicrobioL 132, 2321-2328