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.

Amyloid-beta (A beta) fragments are found in plaques of patients with Alzheimers. Three secretases cleave the amyloid precursor protein, producing multiple A beta fragments that accumulate in the brain and fluids of patients with Alzheimers. A beta peptides are difficult to detect using standard methods because of their small size and multiple isoforms. However, multiple peptide fragments can be detected using a single ProteinChip Array-Based assay. Specific antibodies recognizing various amyloid epitopes are immobilized on a ProteinChip Array. Crude samples, such as tissue lysates, serum, cerebral spinal fluid (CSF), or cell culture media, are applied to the antibody-coated arrays. A beta peptides are specifically retained by the antibody, whereas other sample components are removed by washing. The multiple peptide fragments are detected by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS), which can easily resolve the different fragments because of the corresponding changes in peptide mass.

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