title: Purification of proteins from solutions containing residual host cell proteins via preparative crystallization.
abstract: OBJECTIVE
  To investigate quantitatively and reproducibly a scalable, preparative crystallization method in novel stirred tanks using three different protein solutions containing residual microbial host cell proteins (HCP).
  
  
  RESULTS
  Lysozyme from solutions being spiked with up to 15% host cell proteins (HCP) (corresponding to 176,500 ppm) was crystallized within a 2.4-4.6 h at 93.7% yield using NaCl and glycerol. Lipase was crystallized under comparable conditions using NaCl and a mixture of two polyethylene glycols (PEG). Enhanced green fluorescent protein (eGFP) was overexpressed in E. coli yielding a solution containing 23% target protein. Residual HCP content after pre-treatment was 7-16%. eGFP was crystallized from these solutions within 1.75-4 h at 88.7% step yield using ethanol and the same mixture of two PEG as in the case of lipase. HCP contained in the solvent channels of the protein crystals could be removed by diffusive washing yielding final purities at or above 99%.
  
  
  CONCLUSIONS
  Preparative crystallization can be carried out with fast kinetics and high yields from solutions containing residual impurities and may represent an attractive alternative purification method compared to preparative chromatography, especially at large production scales.
authors.affiliation: Institute of Biochemical Engineering, Technische Universität München, Boltzmannstr. 15, 85748, Garching, Germany, hekmat@lrz.tum.de.
authors.forename: Dariusch
authors.initials: D
authors.lastname: Hekmat
authors.forename: Peter
authors.initials: P
authors.lastname: Breitschwerdt
authors.forename: Dirk
authors.initials: D
authors.lastname: Weuster-Botz
mesh_terms: D001426:Bacterial Proteins / Q000302:isolation & purification; D003460:Crystallization / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005990:Glycerol / Q000737:chemistry; D008049:Lipase / Q000302:isolation & purification*; D009113:Muramidase / Q000302:isolation & purification*; D012965:Sodium Chloride / Q000737:chemistry; D012996:Solutions
chemical_list: D001426:Bacterial Proteins; D012996:Solutions; D012965:Sodium Chloride; D008049:Lipase; D009113:Muramidase; D005990:Glycerol