Application of the Protein Maker as a platform purification system for therapeutic antibody research and development

Geneviève Hélie,Marie Parat,Frédéric Massé,Cory J Gerdts,Thomas P Loisel,Allan Matte

doi:10.1016/j.csbj.2016.06.001

Abstract

Within the research and development environment, higher throughput, parallelized protein purification is required for numerous activities, from small scale purification of monoclonal antibodies (mAbs) and antibody fragments for in vitro and in vivo assays to process development and optimization for manufacturing. Here, we describe specific applications and associated workflows of the Protein Maker liquid handling system utilized in both of these contexts. To meet the requirements for various in vitro assays, for the identification and validation of new therapeutic targets, small quantities of large numbers of purified antibodies or antibody fragments are often required. Reducing host cell proteins (HCP) levels following capture with Protein A by evaluating various wash buffers is an example of how parallelized protein purification can be leveraged to improve a process development outcome. Stability testing under various conditions of in-process intermediates, as an example, the mAb product from a clarified harvest, requires parallelized protein purification to generate concurrent samples for downstream assays. We have found that the Protein Maker can be successfully utilized for small-to-mid scale platform purification or for process development applications to generate the necessary purified protein samples. The ability to purify and buffer exchange up to 24 samples in parallel offers a significant reduction in time and cost per sample compared to serial purification using a traditional FPLC system. By combining the Protein Maker purification system with a TECAN Freedom EVO liquid handler for automated buffer exchange we have created a new, integrated platform for a variety of protein purification and process development applications.

Highlights

Purification of antibodies and antibody fragments are key activities in the generation of critical reagents for various in vivo and in vitro assays as part of biotherapeutic lead identification and process development
Columns were equilibrated in Dulbecco's Phosphate Buffered Saline Solution (DPBS, HyClone Laboratories), the sample applied at the appropriate residence time (1 min for Ni Sepharose Excel or 3 min for MabSelect SuRe or Protein G HP) and a portion of the flow-through fraction collected for subsequent non-reducing SDS-PAGE analysis
Columns were washed with Dulbecco's phosphate buffered saline (DPBS) and bound proteins eluted with two column volumes (CV) of sodium-citrate buffer pH 3.6 (MabSelect SuRe), two CV of 100 mM glycine-HCl pH 2.6 (Protein G HP) or one CV of DPBS with 500 mM imidazole pH 8 (IMAC purification)

Summary

Introduction

Purification of antibodies and antibody fragments are key activities in the generation of critical reagents for various in vivo and in vitro assays as part of biotherapeutic lead identification and process development. Various strategies are available to achieve such purification outcomes, and can involve to various extents both automated and manual methods [1,2]. While parallelized purification methods yielding sub-milligram quantities of pure proteins based on packed columns, 96-well plates containing small quantities of chromatographic resins or ligands immobilized to the surfaces of membranes have been developed, there are relatively. Fewer options available for generating purified quantitates of protein in the intermediate (5–100) milligram scale. A few examples of customized solutions to this problem exist, involving integration of existing purification platforms such as the ÄKTA Purifier with a CETAC autosampler [3], ÄKTA Pure [4] or liquid handling robotics [5] have been reported. While some commercial instruments for purification of small quantities of protein have been developed, such as the QIAcube for purification of His-tagged proteins [7], there are few examples of commercial instruments that can be utilized for platform purification at milligram scale

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