(582bj) Human Fc Receptor Fragment Production for Antibody Drugs Purification By Escherichia Coli

Antibody drugs are recently expected for use in molecularly-targeted therapy. However, their production costs are still high, and therefore innovation of producing technique is necessary. One of the factors that arises the market price of antibody drugs is in purification process, and thus we must develop a low-cost affinity purification method instead of protein A affinity chromatography. Human Fc receptor protein can be used for purification of Antibody drugs, and its partial fragment can be produced by recombinant bacteria, yeast, or mammalian cells. In this study, we use recombinant Escherichia coli, which can be expected to grow faster than yeast or mammalian cells. Moreover, we can use inexpensive medium in E.coli culture compared with that in mammalian cell culture. Furthermore, the scale-up of culture system of E.coli is much easier than that of mammalian cells. The heterologous gene can be introduced into E.coli cells with plasmid vectors. The recombinant E.coli is often used for expression of the heterologous protein.

In this study, we tried to produce Fc receptor fragment in large quantities in order to reduce the cost of purification process in the antibody drugs production. After transformation of E.coli cells with the expression vector for Fc receptor fragment, we cultivate the recombinant E.coli using flask or Jar-fermenter. In the flask culture, E.coli cells grew slowly and produced small amount of affinity ligand because the nutrients in the medium was not enough for long-term cultivation. In the case of the culture using Jar-fermenter, we controlled temperature of culture broth, agitation speed, pH, dissolved oxygen (DO), and feeding rates of nutrients. At the first stage of cultivation for 16h, we carried out a batch culture. After depletion of glucose in the culture medium, we started a fed-batch culture by DO-stat (controlling feeding rate of nutrients based on the value of dissolved oxygen for maintaining optimum culture condition).When E.coli cells efficiently grew in exponential growth phase, the expression of target protein was started by addition of IPTG into the medium. During cultivation, we measured cell growth, glucose concentration, and production amount of Fc receptor fragment.

Consequently, using the Jar-fermenter system, we obtained 123mg/L of target protein, which is 400 times larger than that of the flask culture. We are going to improve and optimize the culture condition for increase in productivity of Fc receptor fragment.