Single‐step affinity purification of enzyme biotherapeutics: A platform methodology for accelerated process development

Abstract

Downstream sample purification for quality attribute analysis is a significant bottleneck in process development for non-antibody biologics. Multi-step chromatography process train purifications are typically required prior to many critical analytical tests. This prerequisite leads to limited throughput, long lead times to obtain purified product, and significant resource requirements. In this work, immunoaffinity purification technology has been leveraged to achieve single-step affinity purification of two different enzyme biotherapeutics (Fabrazyme® [agalsidase beta] and Enzyme 2) with polyclonal and monoclonal antibodies, respectively, as ligands. Target molecules were rapidly isolated from cell culture harvest in sufficient purity to enable analysis of critical quality attributes (CQAs). Most importantly, this is the first study that demonstrates the application of predictive analytics techniques to predict critical quality attributes of a commercial biologic. The data obtained using the affinity columns were used to generate appropriate models to predict quality attributes that would be obtained after traditional multi-step purification trains. These models empower process development decision-making with drug substance-equivalent product quality information without generation of actual drug substance. Optimization was performed to ensure maximum target recovery and minimal target protein degradation. The methodologies developed for Fabrazyme were successfully reapplied for Enzyme 2, indicating platform opportunities. The impact of the technology is significant, including reductions in time and personnel requirements, rapid product purification, and substantially increased throughput. Applications are discussed, including upstream and downstream process development support to achieve the principles of Quality by Design (QbD) as well as integration with bioprocesses as a process analytical technology (PAT).

Reexamining opportunities for therapeutic protein production in eukaryotic microorganisms

“…The lower loads of host cell proteins and lack of mammalian viruses present an opportunity to simplify unit operations and consider new modes of purification that could scale more easily and be operated at lower cost. For example, the large number of chromatography steps required for non‐mAb purification (Brower et al, ) could potentially be reduced if the initial feed has higher purity. If high molecular weight impurities are at low enough concentrations, preparative crystallization of proteins may offer significant economic advantages compared to chromatography (Hekmat, ).…”

End-to-end integrated fully continuous production of recombinant monoclonal antibodies

“…The development scale studies discussed above are meant to serve as a starting point for implementation of an end-to-end continuous biomanufacturing platform. Additional development efforts are required for a reliable and robust manufacturing process, such as at/in-line monitoring of the product quality using Process Analytical Technology (PAT, (Brower et al, 2014;Read et al, 2010;Tharmalingam et al, 2014)), data management solutions, and validation strategies specifically developed for the continuous process paradigm. Enabling technology such as a manufacturing scale PCC and chromatography columns that are functionally closed and capable of operating bioburden-free for prolonged periods of time developed (Warikoo 2014).…”

Analytical Challenges and Advancements in Bioanalysis of Therapeutic Proteins

“…However, these methods also face several challenges including intensive sample preparation prior to analysis, requirement of spectral libraries to determine the structure and composition of analyzed therapeutic protein and in some cases may require high amount of sample and provide limited information about the protein species [14]. Another hyphenation approach, SE-LC is employed for determining the purity, analyzing heterogeneity of charges and some specific post-translational modifications [15]. SE-LC can be further coupled with MS as an indirect interface since direct coupling is limited due to presence of high amount of salts, poor peak shape and impact on protein conformation [8].…”