Recombinant proteins and therapeutic humanized antibodies are widely used in a whole range of disease fields, such as autoimmunity, cancer, inflammation and infectious diseases. In the past, purification of protein and antibody requires specific affinity chromatographic steps. For example, large scale Protein A affinity chromatography is widely used to purify Fc specific antibody such as IgG1, IgG2(code: 5U66). Besides, other immobilized antibody column and immobilized antigen column have been used to separate recombinant protein (rIFN-α) and humanized anti-TAC monoclonal antibody. However, the antigen/antibody interaction of this affinity column for protein /antibody purification was not well understood in 1990’s. In this presentation, protein structure analysis of rIFN-α protein/antibody binding epitope and IL-2 receptor protein /antibody binding interface are studied by x-ray crystallography using the RCSB protein database and Swiss PDF viewer Molecular Model software. 1). Study of structure analysis on interferon-alpha protein binding specificity to neutralizing monoclonal antibody (code:3UX9) was determined by the Molecular Model approach. The molecular level analysis provides structural information on the rIFN-α binding epitope, AB (24-43 AA) and Helix E (140-150 AA), against immunoglobulin Fab site. It provides the structural rationale why the target rIFN-α protein can be specifically removed from cell culture mixtures with this immobilized antibody column. 2).Also, structure analysis of antigen (IL-2 receptor) binding epitope with anti-TAC antibody IgG Fab beta barrel binding site (code: 3NFP) was determined using protein x-ray crystallography and molecular model software. The molecular level analysis provides structural rationale why the anti-TAC antibody can be isolated by its specific binding to the immobilized antigen. This resulting in elution to recover the antibody. In summary, protein structure analysis using protein x-ray crystallography with molecular model software can be used as alternative way to study this immuno-affinity chromatography column binding mechanism for purification of recombinant protein and humanized antibody. 

Structural analysis of large-scale production and purification of recombinant protein and humanized antibody

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.