Antibodies and related proteins comprise one of the largest and fastest-growing classes of protein pharmaceuticals. A majority of such molecules are monoclonal antibodies; however, many new entities are antibody fragments. Due to their structural, physiological, and pharmacological properties, antibody fragments offer new biopharmaceutical opportunities. In the case of recombinant full-length antibodies with suitable Fc regions, two or three column purification processes centered around Protein A affinity chromatography have proven to be fast, efficient, robust, cost-effective, and scalable. Most antibody fragments lack Fc and suitable affinity for Protein A. Adapting proven antibody purification processes to antibody fragments demands different affinity chromatography. Such technology must offer the unit operation advantages noted above, and be suitable for most of the many different types of antibody fragments. Protein L affinity chromatography appears to fulfill these criteria-suggesting its consideration as a key unit operation in antibody fragment processing.
Bioprocess development is a relatively new field for the application of high‐throughput technologies (HTT). Similar to historical applications in drug discovery, these new tools are generally being applied to increase throughput and efficiency to either enable improved performance by testing a larger experimental design space or improve speed for development for biological pipelines. In surveying the general application of these technologies in the biotechnology industry, it becomes clear that HTT have targeted a few key steps within upstream and downstream process development workflows (also called HTPD i.e. high‐throughput process development). The primary focus for upstream HTT has been cell line selection and cell culture development, whereas downstream HTT has centered on the chromatography unit operation. The successful implementation of these technologies has come with many lessons learned on technology selection, workflow validation, and study execution. Furthermore towards improving the application of HTT, analytical techniques in combination with experimental design strategies have been developed that enable larger and more complete scientific investigation of experimental conditions. The complete set of tools, techniques, and design strategies have yielded tangible benefits for early adopters, particularly where capacity and timeline restrictions create workflow bottlenecks. Applied properly, high‐throughput technologies can be an important tool in meeting the bioprocess challenges of today and tomorrow.
In this work a Fab fragment was produced by papain cleavage of a purified monoclonal antibody (Mab) followed by purification on MabSelect™ Sure™ and Capto™ L media (protein A and protein L, respectively, immobilized onto high flow agarose). Fractions from the different steps were analyzed by gel filtration (GF) to follow purification progress. The process started with Mab being purified from feed on MabSelect Sure and thereafter the Mab was digested by papain overnight at 37 degrees into Fab‐ and Fcfragments. Cleavage was interrupted with the inhibitor antipain and the digest was applied to a column containing MabSelect Sure. Fc and partially digested Mab bind to the chromatography medium whereas the Fab fragment elutes in the flow through together with other contaminants such as papain. The flow through fraction was applied to Capto L medium and the Fab fragment was thus further purified. The purity of the Fab fragment was confirmed by analysis on new high resolution GF media. This new GF media has the resolution power to separate both Mab from aggregates and Fab fragments from Fc fragments.
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