Leveraging high-throughput purification to accelerate viral vector process development.

Recombinant adeno-associated virus (AAV) has been broadly used as a delivery tool for gene therapy applications. The development of a robust purification process is essential for delivering high purity and quality AAV products to clinic. The short clinical timelines and material limitations of early-stage development pose unique challenges to developing robust and scalable downstream purification processes. One approach to overcome these limitations is to leverage high throughput (HTP) strategies and automation technologies for purification process development, an approach that is well established in protein biologics and other areas. However, due to the unique challenges related to viral vector purification, implementing HTP approaches for gene therapy process development has not been explored extensively. In this paper, we established a HTP chromatography platform and demonstrated its capability to facilitate gene therapy purification process development using both mini-columns and self-packed resin plates. The end-to-end development workflow for AAV HTP purification is detailed in this work with the expectation of serving as an introductory for the AAV purification development field. Comparable process performance was confirmed between a bench-scale chromatography process and an HTP chromatography format. Slightly lower recovery was observed using the HTP format (62% vs 75%), as well as %full capsid enrichment (71% vs. 82%). Comparable impurity clearance capability was demonstrated between the two different systems as well. It was concluded that the established HTP chromatography formats can serve as a surrogate to bench-scale chromatography development to reduce material needs and development timelines for AAV purification development.

Separating Empty and Full Recombinant Adeno-Associated Virus Particles Using Isocratic Anion Exchange Chromatography.

The development of recombinant adeno-associated virus (rAAV) gene therapies is becoming an increasing priority in the biotherapeutic landscape. One of the challenges associated with the production of rAAV is the formation of empty AAV particles that do not contain a therapeutic gene. The concerns about the impact of empty particles on clinical safety and rAAV-mediated gene expression have necessitated the development of purification processes to remove these species. The development of a robust and scalable purification process to separate empty and full AAV particles at large scale remains a challenge. In this study, a novel anion exchange chromatography process based on isocratic wash and elution steps to enrich full rAAV2 particles is presented. An operating design space is identified to ensure the robustness of the process. The isocratic chromatography provides several advantages over the traditional shallow linear gradient elution, including lower buffer consumption, smaller intermediate pool volumes, and more robust manufacturing.

Analytical Strategies for Quantification of Adeno-Associated Virus Empty Capsids to Support Process Development.

Recombinant adeno-associated virus (rAAV)-mediated gene therapy is a fast-evolving field in the biotechnology industry. One of the major challenges in developing a purification process for AAV gene therapy is establishing an effective yet scalable method to remove empty capsids, or viral vectors lacking the therapeutic gene, from full capsids-viral product containing the therapeutic sequence. Several analytical methods that can quantify the empty-to-full capsid ratio have been reported in the literature. However, as samples can vary widely in viral titer, buffer matrix, and the relative level of empty capsids, understanding the specifications and limitations of different analytical methods is critical to providing appropriate support to facilitate process development. In this study, we developed a novel anion-exchange high-performance liquid chromatography assay to determine the empty-to-full capsid ratio of rAAV samples. The newly developed method demonstrated good comparability with both the transmission electron microscopy and analytical ultracentrifugation methods used in empty-to-full capsid ratio quantification, while providing much higher assay throughput and reducing the minimum sample concentration requirement to 2.7E11 viral genomes/mL.

Leveraging single-pass tangential flow filtration to enable decoupling of upstream and downstream monoclonal antibody processing.

Decoupling upstream and downstream operations in biopharmaceutical production could enable more flexible manufacturing operations and could allow companies to leverage strategic or financial benefits that would be otherwise unattainable. A decoupling process was developed and scaled up utilizing single-pass tangential flow filtration for volume reduction, followed by bulk freezing in single-use bags prior to purification. Single-pass tangential flow filtration can be used to continuously concentrate harvested cell culture fluid, reducing the volume by 15-25× with a step yield of >96%. These concentration factors were reproduced with a second product, indicating that the process could be amenable to platform processes. Experimental data indicate that the product tested was stable for at least one year at -40 or -70°C. The concentration of the harvested cell culture fluid-either with or without a subsequent period of frozen storage-had no impact on the product quality attributes that were tested. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:405-411, 2018.

Leveraging a CHO cell line toolkit to accelerate biotherapeutics into the clinic.

The Biogen upstream platform is capable of delivering equivalent quality material throughout the cell line generation process. This allows us to rapidly deliver high-quality biopharmaceuticals to patients with unmet medical needs. The drive to reduce time-to-market led the cell engineering group to develop an expression system that can enable this strategy. We have developed a clonal Chinese Hamster Ovary (CHO) host cell line that can routinely produce consistent antibody material at high titers throughout the cell line generation process. This host line enables faster delivery of early phase material through use of the highly productive stable pool or a mixture of high performance clones. Due to unique characteristics of this cell line, the product quality of material from early cell populations is very comparable to material from the final clones. This lends itself to a "fast-to-tox" strategy whereby toxicology studies can be performed with representative material from an earlier cell population, thus accelerating the clinical timelines. Our new clonal host offers robust and consistent performance that enables a highly productive, flexible process and faster preclinical timelines. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1468-1475, 2017.

Evaluation of eco-friendly zwitterionic detergents for enveloped virus inactivation.

Inclusion of a detergent in protein biotherapeutic purification processes is a simple and very robust method for inactivating enveloped viruses. The detergent Triton X-100 has been used for many years and is part of the production process of several commercial therapeutic proteins. However, recent ecological studies have suggested that Triton X-100 and its break-down products can potentially behave as endocrine disrupters in aquatic organisms, raising concerns from an environmental impact perspective. As such, discharge of Triton X-100 into the waste water treatment plants is regulated in some jurisdictions, and alternative detergents for viral inactivation are required. In this work, we report on the identification and evaluation of more eco-friendly detergents as viable replacements for Triton X-100. Five detergent candidates with low to moderate environmental impact were initially identified and evaluated with respect to protein stability, followed by proof-of-concept virus inactivation studies using a model enveloped virus. From the set of candidates lauryldimethylamine N-oxide (LDAO) was identified as the most promising detergent due to its low ecotoxicity, robust anti-viral activity (LRV >4 at validation set-point conditions with X-MuLX), and absence of any negative impact on protein function. This detergent exhibited effective and robust virus inactivation in a broad range of protein concentrations, solution conductivities, pHs, and in several different cell culture fluid matrices. The only process parameter which correlated with reduced virus inactivation potency was LDAO concentration, and then only when the concentration was reduced to below the detergent's critical micelle concentration (CMC). Additionally, this work also demonstrated that LDAO was cleared to below detectable levels after Protein A affinity chromatography, making it suitable for use in a platform process that utilizes this chromatographic mode for protein capture. All these findings suggest that LDAO may be a practical alternative to Triton X-100 for use in protein therapeutic production processes for inactivating enveloped viruses. Biotechnol. Bioeng. 2017;114: 813-820. © 2016 Wiley Periodicals, Inc.

Pool-less processing to streamline downstream purification of monoclonal antibodies.

With cell culture titers and productivity increasing in the last few years, pressure has been placed on downstream purification to look at alternative strategies to meet the demand of biotech products with high dose requirements. Even when the upstream process is not continuous (perfusion based), adopting a more productive and/or continuous downstream process can be of significant advantage. Due to the recent trend in exploring continuous processing options for biomolecules, several enabling technologies have been assessed at Biogen. In this paper, we evaluate the capability of one of these technologies to streamline and improve our downstream mAb purification platform. Current conventional downstream polishing steps at Biogen are operated in flow-through mode to achieve higher loadings while maintaining good selectivity. As titers increase, this would result in larger columns and larger intermediate product pool holding tanks. A semicontinuous downstream process linking the second and third chromatography steps in tandem can reduce/eliminate intermediate holding tanks, reduce overall processing time, and combine unit operations to reduce validation burdens. A pool-less processing technology utilizing inline adjustment functionality was evaluated to address facility fit challenges for three high titer mAbs. Two different configurations of polishing steps were examined: (i) anion exchange and hydrophobic interaction and (ii) anion exchange and mixed mode chromatography. Initial laboratory scale proof of concept studies showed comparable performance between the batch purification process and the pool-less process configuration.

Retrospective Evaluation of Low-pH Viral Inactivation and Viral Filtration Data from a Multiple Company Collaboration.

Considerable resources are spent within the biopharmaceutical industry to perform viral clearance studies, which are conducted for widely used unit operations that are known to have robust and effective retrovirus clearance capability. The collaborative analysis from the members of the BioPhorum Development Group Viral Clearance Working Team considers two common virus reduction steps in biopharmaceutical processes: low-pH viral inactivation and viral filtration. Analysis included eight parameters for viral inactivation and nine for viral filtration. The extensive data set presented in this paper provides the industry with a reference point for establishing robust processes in addition to other protocols available in the literature (e.g., ASTM Std. E2888-12 for low-pH inactivation). In addition, it identifies points of weakness in the existing data set and instructs the design and interpretation of future studies. Included is an abundance of data that would have been difficult to generate individually but collectively will help support modular viral clearance claims.

High-throughput ion exchange purification of positively charged recombinant protein in the presence of negatively charged dextran sulfate.

Product quality analyses are critical for developing cell line and bioprocess producing therapeutic proteins with desired critical product quality attributes. To facilitate these analyses, a high-throughput small-scale protein purification (SSP) is required to quickly purify many samples in parallel. Here we develop an SSP using ion exchange resins to purify a positively charged recombinant growth factor P1 in the presence of negatively charged dextran sulfate supplemented to improve the cell culture performance. The major challenge in this work is that the strong ionic interaction between P1 and dextran sulfate disrupts interaction between P1 and chromatography resins. To solve this problem, we develop a two-step SSP using Q Sepharose Fast Flow (QFF) and SP Sepharose XL (SPXL) resins to purify P1. The overall yield of this two-step SSP is 78%. Moreover, the SSP does not affect the critical product quality attributes. The SSP was critical for developing the cell line and process producing P1.

Applying high-throughput methods to develop a purification process for a highly glycosylated protein.

Micro-scale chromatography formats are becoming more routinely used in purification process development because of their ability to rapidly screen large number of process conditions at a time with minimal material. Given the usual constraints that exist on development timelines and resources, these systems can provide a means to maximize process knowledge and process robustness compared to traditional packed column formats. In this work, a high-throughput, 96-well filter plate format was used in the development of the cation exchange and hydrophobic interaction chromatography steps of a purification process designed to alter the glycoform distribution of a small protein. The significant input parameters affecting process performance were rapidly identified for both steps and preliminary operating conditions were identified. These ranges were verified in a packed chromatography column in order to assess the ability of the 96-well plate to predict packed column performance. In both steps, the 96-well plate format consistently led to underestimated glycoform-enrichment levels and to overestimated product recovery rates compared to the column-based approach. These studies demonstrate that the plate format can be used as a screening tool to narrow the operating ranges prior to further optimization on packed chromatography columns.

Modeling industrial centrifugation of mammalian cell culture using a capillary based scale-down system.

Continuous-flow centrifugation is widely utilized as the primary clarification step in the recovery of biopharmaceuticals from cell culture. However, it is a challenging operation to develop and characterize due to the lack of easy to use, small-scale, systems that can be used to model industrial processes. As a result, pilot-scale continuous centrifugation is typically employed to model large-scale systems requiring a significant amount of resources. In an effort to reduce resource requirements and create a system which is easy to construct and utilize, a capillary shear device, capable of producing energy dissipation rates equivalent to those present in the feed zones of industrial disk stack centrifuges, was developed and evaluated. When coupled to a bench-top, batch centrifuge, the capillary device reduced centrate turbidity prediction error from 37% to 4% compared to using a bench-top centrifuge alone. Laboratory-scale parameters that are analogous to those routinely varied during industrial-scale continuous centrifugation were identified and evaluated for their utility in emulating disk stack centrifuge performance. The resulting relationships enable bench-scale process modeling of continuous disk stack centrifuges using an easily constructed, scalable, capillary shear device coupled to a typical bench-top centrifuge.

Downstream antibody purification using aqueous two-phase extraction.

The extraction of antibodies using a polyethylene glycol (PEG)-citrate aqueous two-phase system (ATPS) was investigated. Studies using purified monoclonal antibody (mAb) identified operating ranges for successful phase formation and factors that significantly affected antibody partitioning. The separation of antibody and host cell protein (HCP) from clarified cell culture media was examined using statistical design of experiments (DOE). The partitioning of antibody was nearly complete over the entire range of the operating space examined. A model of the HCP partitioning was generated in which both NaCl and citrate concentrations were identified as significant factors. To achieve the highest purity, the partitioning of HCP from cell culture fluid into the product containing phase was minimized using a Steepest Descent algorithm. An optimal ATPS consisting of 14.0% (w/w) PEG, 8.4% (w/w) citrate, and 7.2% (w/w) NaCl at pH 7.2 resulted in a product yield of 89%, an approximate 7.6-fold reduction in HCP levels relative to the clarified cell culture fluid before extraction and an overall purity of 70%. A system consisting of 15% (w/w) PEG, 8% (w/w) citrate, and 15% (w/w) NaCl at pH 5.5 reduced product-related impurities (aggregates and low molecular product fragments) from ∼40% to less than 0.5% while achieving 95% product recovery. At the experimental conditions that were optimized in the batch mode, a scale-up model for the use of counter-current extraction technology was developed to identify potential improvements in purity and recovery that could be realized in the continuous operational mode.

Using partition designs to enhance purification process understanding.

Characterization of purification processes by identifying significant input parameters and establishing predictive models is vital to developing robust processes. Current experimental design approaches restrict analysis to one process step at a time, which can severely limit the ability to identify interactions between process steps. This can be overcome by the use of partition designs which can model multiple, sequential process steps simultaneously. This paper presents the application of partition designs to a monoclonal antibody purification process. Three sequential purification steps were modeled using both traditional experimental designs and partition designs and the results compared as a proof of concept study. The partition and traditional design approaches identified the same input parameters within each process step that significantly affected the product quality output examined. The partition design also identified significant interactions between input parameters across process steps that could not be uncovered by the traditional approach.