Production Process Scale-up Feasibility Analysis Of Humanized Engineerd Antibody Anti-HBsAg Fab In Prokaryotic Expression System

Objective:1. To practice the multi-variable analysis of the factors that may affect the growth and the foreign protein expression of engineered E.coli in shake-flask scale and confirm how these factors affect the growth and protein expression.2. To explore the high-cell density cultivation methods in BioFlollo fermentor(NBS company) to buildup a feasible fermentation routine.3.. To explore the antibody fragment purification technics that is befitting for production scale.Methods:1. We first ascertain the eight need-to-exploring factors that may affect the growth and protein expression of E.coli by pre-experiments and the literature reviews. Then we design the experiments to test how these factors work with the corresponding statistics analysis methods-Plackett-Burman multi-variable-two-level method and Box-Behnken response surface analysis method.2. In BioFllo fermentor, we explore the three fed-batch fermentation methods---constant feeding fed-batch strategy, exponential feeding fed-batch strategy and DO-stat feeding fed-batch strategy---to see what fermentation level these feeding strategies can achieve.3. In the steps of designing purification technics, we first ensure the purification condition of the cation-exchange chromatography and hydrophobic interaction chromatography with the purified Fab(by Ni2+affinity chromatography), and then purify the prepared freezing-and-thawing supemant of E.coli according the combination chromatography strategy---cation-exchange chromatography, hydrophobic interaction chromatography and gelfiltration chromatography. At last, we mensurate the functional affinity-5-constant of Fab with non-competition ELISA method.Results:1. The r esults o f m ulti-variable a nalysis o f factors which c ould a ffect the E.coli growth show the consequential sequence of these eight factors and reckon the pH value and trace element content as the most important two factors. The RSA (responses surface analysis) results of factors that could affect the foreign protein expression in E.coli show that the concentration and induction time of inductor and the interaction effect of these two factor are the most important three factors.2. The analysis results of fed-batch fermentation strategy which include constant feeding fed-batch fermentation, exponential feeding fed-batch fermentation and DO-stat feeding fed-batch fermentation show that although the highest mass of E.coli and highest quantity of Fab expressed can be abtained by DO-stat method, the highest Fab productivity of time and mass unit can only be abtained by constant feeding fed-batch method. The best fermentation method seems to be the constant feeding fed-batch fermentation strategy according to the BioFlo 110 fermentor equipment in our lab.3. The results of combination chromatography purification strategy demonstrate the feasibility of purifying the antibody fragment in production scale, and the purification efficient is about 65% with our purification method. The functional affinity constant of Fab proves to be about 10-10M~ only about less than 100 times when comparing with the integrity antibody anti-HBsAg IgGConclusion:We have confirmed the consequential factors that can affect the growth and protein expression in E.coli in shake-flask scale; have compared thefermentation results of the three fed-bath fermentation methods and brought-6-forward the ideal fermentation routine in our BioFlo 110 fermentor; and have figured out the combination chromatography purification technic for Fab.We demonstrated the feasibility of producing antibody fragment in production scale according our experiments results.