Rapid optimization of antibotulinum toxin antibody fragment production by an integral approach utilizing RC-SELDI mass spectrometry and statistical design.
A process for the rapid development and optimization of the fermentation process for an antibotulinum neurotoxin antibody fragment (bt-Fab) production expressed in Escherichia coli was achieved via a high-throughput process proteomics and statistical experimental design. This process, using retentate chromatography-surface enhanced laser desorption/ionization mass spectrometry (RC-SELDI MS), was employed for identifying and quantifying bt-Fab antibody in complex biological samples for the optimization of microbial fermentation conditions. Five variables (type of culture media, glycerol concentration, post-induction temperature, IPTG concentration, and incubation time after induction) were statistically combined using an experimental 2(5)(-1) fractional factorial design and tested for their effects on maximal bt-Fab antibody production. When the effects of individual variables and their interactions were assessed, type of media and post-induction temperature showed statistically significant increase in yield of the fermentation process for the maximal bt-Fab antibody production. This study establishes an integral approach as a valuable tool for the rapid development of manufacturing processes for producing various biological materials. To verify the RC-SELDI MS method, a Fab-specific immuno-affinity HPLC assay developed here was also employed for the quantification of the bt-Fab antibody in crude lysate samples obtained during the fermentation optimization process. Similar results were obtained.
Park
Jun T
JT
U.S. Army Edgewood Chemical Biological Center, Aberdeen Proving Ground, Maryland, USA. jun.park@us.army.mil
Bradbury
Lisa
L
Kragl
Frank J
FJ
Lukens
Dennis C
DC
Valdes
James J
JJ
D001905:Botulinum Toxins / Q000276:immunology*; D002851:Chromatography, High Pressure Liquid; D003470:Culture Media; D004926:Escherichia coli / Q000378:metabolism; D005285:Fermentation; D006358:Hot Temperature; D007140:Immunoglobulin Fab Fragments / Q000032:analysis / Q000096:biosynthesis*; D007218:Industrial Microbiology / Q000379:methods; D012107:Research Design / Q000706:statistics & numerical data*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013997:Time Factors
D003470:Culture Media; D007140:Immunoglobulin Fab Fragments; D001905:Botulinum Toxins