Rapid Optimization of Antibotulinum Toxin Antibody Fragment Production by an Integral Approach Utilizing RC-SELDI Mass Spectrometry and Statistical Design

Abstract

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.

Strategies for producing recombinant sucrose phosphorylase originating from Bifidobacterium longum in Escherichia coli JM109

“…In induction temperature experiments, many studies have drawn different conclusions from that of our study [13,[25][26][27][28][29][30]. These studies examined recombinant protein production by varying the induction temperature between 18 8C and 45 8C, and claimed that induction at 20-30 8C was more efficient for obtaining more soluble protein formation than at 37 8C.…”

Mass spectrometry to describe product and contaminant adsorption properties for bioprocess development

“…The majority of SELDI‐MS publications relate to its use in biomarker discovery. However, other published applications include fermentation/cell‐culture optimization (Mukhopadhyay et al, 2005; Park et al, 2006), process monitoring (Brenac et al, 2008; Weinberger et al, 2002), purification development (Merchant and Weinberger, 2000), and product analysis (Weinberger et al, 2002; Woolley and Al Rubeai, 2009). Other groups have also used SELDI‐MS to evaluate purification steps by using it to analyze column fractions (Fortis et al, 2006; Guerrier et al, 2005, 2007; Wierling et al, 2007) and a study by Santambien and colleagues, used SELDI ProteinChips to predict the chromatography step for endostatin purification from Pichia pastoris .…”

Product and contaminant measurement in bioprocess development by SELDI‐MS

“…There are many publications discussing the background of SELDI‐MS and how it works 5–9. The majority of published material involving the use of SELDI‐MS has been related to biomarker discovery, but there is also literature on its use in fermentation/cell‐culture optimization,10, 11 purification development,5 process monitoring,8, 12 and product analysis 4, 8. For fermentation it has been used to measure an antibotulinum neurotoxin antibody fragment (bt‐Fab) 11.…”