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Biopharmaceutical separations require tremendous amounts of optimization to achieve acceptable product purity. Typically, large volumes of reagents and biological materials are needed for testing different parameters, thus adding to the expense of biopharmaceutical process development. This study demonstrates a versatile and customizable microscale column (µCol) for biopharmaceutical separations using immobilized metal affinity chromatography (IMAC) as an example application to identify key parameters. µCols have excellent precision, efficiency, and reproducibility, can accommodate any affinity, ion-exchange or size-exclusion-based resin and are compatible with any high-performance liquid chromatography (HPLC) system. µCols reduce reagent amounts, provide comparable purification performance and high-throughput, and are easy to automate compared with current conventional resin columns. We provide a detailed description of the fabrication methods, resin packing methods, and µCol validation experiments using a conventional HPLC system. Finite element modeling using COMSOL Multiphysics was used to validate the experimental performance of the µCols. In this study, µCols were used for improving the purification achieved for granulocyte colony stimulating factor (G-CSF) expressed using a cell-free CHO in vitro translation (IVT) system and were compared to a conventional 1 ml IMAC column. Experimental data revealed comparable purity with a 10-fold reduction in the amount of buffer, resin, and purification time for the μCols compared with conventional columns for similar protein yields., (© 2018 Wiley Periodicals, Inc.)

Chromatofocusing is investigated as an alternative to protein A chromatography for the initial capture step in a purification process for several monoclonal antibodies and antibody fusion products. For comparison, this work also investigates the use of ion-exchange chromatography with either pH or salt gradient elution as additional alternatives to protein A chromatography. The specific conditions employed for the capture step for the case of chromatofocusing were selected on a rational basis using a computer-aided design method implemented in the form of a Microsoft Excel spreadsheet. Alternative operating conditions were compared experimentally with regard to the product yield achieved as well as the removal of total host cell proteins (HCPs) and of a specific HCP major component. Results from this study indicate that both chromatofocusing and ion-exchange chromatography are useful alternatives to a protein A chromatography capture step in many practical cases. This is especially true for the case of chromatofocusing when it is possible to exploit the ability of the method to create complex gradient shapes that are self-forming inside the column and to simultaneous focus and separate proteins inside the column., (Copyright © 2018 Elsevier B.V. All rights reserved.)