Comparison of the Escherichia coli proteomes for recombinant human growth hormone producing and nonproducing fermentations.
Two-dimensional electrophoretic analyses of Escherichia coli cells producing recombinant human growth hormone (Nutropin) in fermentations were conducted. The resulting two-dimensional protein profiles were compared with those of nonproducing (blank) cells. A qualitative comparison was performed to address regulatory issues in the biopharmaceutical industry, and a semiquantitative comparison was performed to reveal information about the physiological state of the cells. The protein spots unique to production fermentation profiles were all related to recombinant human growth hormone (hGH); these included intact hGH, charge variants of hGH, and a proteolytically cleaved form of hGH, as expected. There were no E. coli host cell proteins unique to either the production or blank fermentation profiles. Rather, all detectable differences in E. coli proteins were quantitative in nature. Specifically, the levels of IbpA (inclusion body binding protein A), Ivy (inhibitor of vertebrate lysozyme), and a cleaved form of GroEL (Hsp60 homolog) were higher in hGH production profiles, whereas the levels of GlmU protein and PspA (phage shock protein A) were higher in blank profiles. In general, the high degree of similarity between proteomes for hGH-producing and nonproducing cells suggests that E. coli proteins from a nonproducing (blank) fermentation are appropriate for eliciting antibodies that are then used in immunoassays to measure host cell proteins in samples from production fermentations.
Champion
Kathleen M
KM
Department of Analytical Chemistry, Genentech Inc., MS #62, 1 DNA Way, South San Francisco, CA 94080, USA. kmc@gene.com
Nishihara
Julie C
JC
Aldor
Ilana S
IS
Moreno
G Tony
GT
Andersen
Dana
D
Stults
Kathy L
KL
Vanderlaan
Martin
M
D000465:Algorithms; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D019149:Bioreactors; D002352:Carrier Proteins / Q000378:metabolism; D018834:Chaperonin 60 / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism; D005285:Fermentation; D006360:Heat-Shock Proteins / Q000378:metabolism; D019382:Human Growth Hormone / Q000096:biosynthesis*; D006801:Humans; D007118:Immunoassay; D015151:Immunoblotting; D010957:Plasmids / Q000378:metabolism; D020543:Proteome; D011994:Recombinant Proteins / Q000378:metabolism*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D018834:Chaperonin 60; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; C097578:IbpA protein, E coli; C429555:Ivy protein, E coli; D020543:Proteome; D011994:Recombinant Proteins; C069816:phage shock protein, Bacteria; D019382:Human Growth Hormone