Abstract
We demonstrate here a rapid alternative method for the production of functional bi-specific antibodies using the mild reducing agent 2-mercaptoethanesulfonic acid sodium salt (MESNA). Following reduction of a mixture of two monoclonal antibodies with MESNA to break inter heavy chain bonds, this solution is dialysed under oxidising conditions and antibodies are allowed to reform. During this reaction a mixture of antibodies is formed, including parental antibodies and bi-specific antibody. Bi-specific antibodies are purified over two sequential affinity columns. Following purification, bi-specificity of antibodies is determined in enzyme-linked immunosorbent assays and by flow cytometry. Using this redox method we have been successful in producing hybrid and same-species bi-specific antibodies in a time frame of 6–10 working days, making this production method a time saving alternative to the time-consuming traditional heterohybridoma technology for the production of bi-specific antibodies for use in early pilot studies. The use of both rat and mouse IgG antibodies forming a rat/mouse bi-specific antibody as well as producing a pure mouse bi-specific antibody and a pure rat bi-specific antibody demonstrates the flexibility of this production method.
Highlights
Kohler and Milstein [1] pioneered hybridoma technology and thereby opened the possibility to manufacture pure monoclonal antibody in large amounts
We have developed a chemical reduction-oxidation method for the production of purified bi-specific antibodies (bsAb) in a fraction of the time taken by the traditional hybrid hybridoma technology by using the mild reducing agent 2-mercaptoethanesulfonic acid sodium salt (MESNA) followed by dialysis under oxidising conditions in order to allow antibodies to reform
Production of same species bsAbs In order to be able to purify bi-specific antibodies made from the same species and antibody subclass, parent antibodies were pre-labeled with biotin or DNP prior to redox exposure
Summary
Kohler and Milstein [1] pioneered hybridoma technology and thereby opened the possibility to manufacture pure monoclonal antibody (mAb) in large amounts. We have developed a chemical reduction-oxidation (redox) method for the production of purified bsAbs in a fraction of the time taken by the traditional hybrid hybridoma technology by using the mild reducing agent 2-mercaptoethanesulfonic acid sodium salt (MESNA) followed by dialysis under oxidising conditions in order to allow antibodies to reform.
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