Abstract
Affinity chromatography provides an excellent platform for protein purification, which is a key step in the large scale downstream processing of therapeutic monoclonal antibodies (Mabs). Protein A chromatography constitutes the gold standard for Mab purification. However, the required acidic conditions (2.8–3.5) for elution from the affinity matrix limit their applicability, particularly for next generation antibodies and antibody fusion proteins, since denaturation and irreversible aggregation can occur due to the acidic buffer conditions. Here we describe a generic procedure for the generation of antigen-specific chromatography ligands with tailor-made elution conditions. To this end, we generated a scFv-library based on mRNA from a chicken immunized with human Fc. The antibody repertoire was displayed on yeast Saccharomyces cerevisiae screened via FACS toward pH- and magnesium-responsive scFvs which specifically recognize human IgG antibodies. Isolated scFvs were reformatted, produced in Escherichia coli and immobilized on NHS-agarose columns. Several scFvs were identified that mediated antibody binding at neutral pH and antibody recovery at pH values of 4.5 and higher or even at neutral pH upon MgCl2 exposure. The iterative screening methodology established here is generally amenable to the straightforward isolation of stimulus-responsive antibodies that may become valuable tools for a variety of applications.
Highlights
In the time period of 2014 to 2018 a total of 129 unique biopharmaceuticals were approved for either the US or EU market raising the total number of approved biopharmaceuticals to 316
We show that the combination of avian immunization, yeast surface display (YSD) and ultra-high throughput screening by FACS results in the isolation of numerous human Fc-specific pH- and magnesium-responsive single chain fragment variable (scFv)
We describe the fast and straightforward generation of chicken derived antibody fragments which can be used as ligands in affinity chromatography
Summary
In the time period of 2014 to 2018 a total of 129 unique biopharmaceuticals were approved for either the US or EU market raising the total number of approved biopharmaceuticals to 316. The category of biopharmaceuticals contains monoclonal antibodies (mAbs), hormones, clotting factors, enzymes and others. This heterogeneous group of molecules is mainly utilized in cancer, inflammation-related, hemophilia and diabetes indications and is responsible for $188 bn sales. With up to 65.5% of total sales, monoclonal antibodies remain the most interesting protein scaffold. The market became more congested and multiple molecules are utilized for similar indications. This leads to an increase in competitive pressure, a motivation for research and development of new low-cost production and purification strategies (Walsh, 2018)
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