Impact of clarification strategy on chromatographic separations: Pre‐processing of cell homogenates

This research focused on how the extent and type of primary solid–liquid separation can affect the performance of guard filtration and chromatography, in this instance hydrophobic interaction chromatography. The system used in the study was yeast (Saccharomyces cerevisiae) with the target molecule being an intracellular protein; alcohol dehydrogenase (ADH). As expected, loading more poorly clarified suspensions (both centrates and primary filtrates) required proportionally larger guard filtration areas. In addition for feed suspensions prepared by centrifugation, increased clarification led to greater column capacity. However, where filtration was used to achieve similar clarification considerably lower column capacity was achieved. These results were attributed to centrifugation leading to the aggregation of lipids and their subsequent removal in this form before application to the column. Clarification by filtration leaves such lipids in their original “soluble” state and hence they are not removed. The importance of the need to examine such interactive effects in bioprocess studies is discussed. This observation was confirmed with further analytical work into the nature of the aggregated material formed in the supernatant under centrifugation conditions. This material was only soluble in an organic solvent, and identified as phophatidylcholine and ergosterol as among the components removed by centrifugation and guard filtration as opposed to filtration and guard filtration. Biotechnol. Bioeng. 2008;100: 941–949. © 2008 Wiley Periodicals, Inc.

[1]  T. Gerngross,et al.  Advances in the production of human therapeutic proteins in yeasts and filamentous fungi , 2004, Nature Biotechnology.

[2]  K. Jansen,et al.  Purification of virus-like particles of recombinant human papillomavirus type 11 major capsid protein L1 from Saccharomyces cerevisiae. , 1999, Protein expression and purification.

[3]  C. Horváth,et al.  Salt effect on hydrophobic interactions in precipitation and chromatography of proteins: an interpretation of the lyotropic series. , 1977, Archives of biochemistry and biophysics.

[4]  N. Titchener-Hooker,et al.  Visualising fouling of a chromatographic matrix using confocal scanning laser microscopy , 2006, Biotechnology and bioengineering.

[5]  Y. K. Chang,et al.  Development of an expanded bed technique for an affinity purification of G6PDH from unclarified yeast cell homogenates. , 1995, Biotechnology and bioengineering.

[6]  M Hoare,et al.  Scale‐down of continuous filtration for rapid bioprocess design: Recovery and dewatering of protein precipitate suspensions , 2003, Biotechnology and bioengineering.

[7]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[8]  A. Bentham,et al.  The separation of affinity flocculated yeast cell debris using a pilot‐plant scroll decanter centrifuge , 1990, Biotechnology and bioengineering.

[9]  Nigel J. Titchener-Hooker,et al.  Effect of fouling on the capacity and breakthrough characteristics of a packed bed ion exchange chromatography column , 2006, Bioprocess and biosystems engineering.

[10]  J. Huddleston,et al.  Influence of system and molecular parameters upon fractionation of intracellular proteins from Saccharomyces by aqueous two-phase partition. , 1991, Enzyme and microbial technology.

[11]  Marie-Françoise Jourdan,et al.  New test method for evaluation of water cartridge filters , 1994 .

[12]  Richard C. Willson,et al.  Purification of plasmid DNA using selective precipitation by compaction agents , 1999, Nature Biotechnology.

[13]  R. Holdich,et al.  Solid-Liquid Filtration and Separation Technology: Rushton/Solid-Liquid , 2008 .

[14]  R Hjorth,et al.  Hydrophobic interaction ligand selection and scale-up of an expanded bed separation of an intracellular enzyme from Saccharomyces cerevisiae. , 2002, Journal of chromatography. A.

[15]  M. Boychyn,et al.  Performance prediction of industrial centrifuges using scale-down models , 2004, Bioprocess and biosystems engineering.

[16]  M. Bard,et al.  Biochemistry, cell biology and molecular biology of lipids of Saccharomyces cerevisiae , 1998, Yeast.

[17]  P Dunnill,et al.  Selective flocculation of nucleic acids, lipids, and colloidal particles from a yeast cell homogenate by polyethyleneimine, and its scale-up. , 1990, Enzyme and microbial technology.

[18]  Gary J. Lye,et al.  An automated microscale technique for the quantitative and parallel analysis of microfiltration operations , 2006 .

[19]  N Hutchinson,et al.  Shear stress analysis of mammalian cell suspensions for prediction of industrial centrifugation and its verification , 2006, Biotechnology and bioengineering.

[20]  M. J. van der Merwe,et al.  Quantification of plasma membrane ergosterol of Saccharomyces cerevisiae by direct-injection atmospheric pressure chemical ionization/tandem mass spectrometry. , 2001, Analytical biochemistry.

[21]  H. Ngo,et al.  An enhancement of critical flux in crossflow microfiltration with a pretreatment of floating medium flocculator/prefilter , 1997 .

[22]  B. Johansson,et al.  Column lifetime of a new agarose medium for high-performance gel filtration chromatography at basic pH. , 1985, Journal of chromatography.

[23]  M. Lilly,et al.  The clarification of mechanically disrupted yeast suspensions by rotary vacuum precoat riltration , 1973 .

[24]  Nigel J. Titchener-Hooker,et al.  Confocal imaging of chromatographic fouling under flow conditions , 2007 .

[25]  P. Dunnill,et al.  The performance of a scaled down industrial disc stack centrifuge with a reduced feed material requirement , 1998 .

[26]  R. Schneiter,et al.  Electrospray Ionization Tandem Mass Spectrometry (Esi-Ms/Ms) Analysis of the Lipid Molecular Species Composition of Yeast Subcellular Membranes Reveals Acyl Chain-Based Sorting/Remodeling of Distinct Molecular Species En Route to the Plasma Membrane , 1999, The Journal of cell biology.