John Pieracci

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. Bioeng. 2011; 108:989–998.

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.

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. Biotechnol. Bioeng. 2010;107: 814–824.

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.

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.

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.

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.

Erratum for John Mattila, Mike Clark, Shengjiang Liu, et al.: “Retrospective Evaluation of Low-pH Viral Inactivation and Viral Filtration Data from a Multiple Company Collaboration”

Ref: Retrospective Evaluation of Low-pH Viral Inactivation and Viral Filtration Data from a Multiple Company Collaboration John Mattila, Mike Clark, Shengjiang Liu, et al. PDA J Pharm Sci and Tech 2016, 70 293-299
Access the most recent version at doi:10.5731/pdajpst.2016.006478 Due to a mathematical error we would like to submit an erratum for figure 6 p298.

Industry Review of Cell Separation and Product Harvesting Methods

Abstract Once a therapeutic protein has been produced by a chosen production system, the product must be separated and recovered from the host system. Cell harvest and recovery serve this function by removing or isolating host cells so that the product can be recovered from the host system, and the product stream can be clarified prior to the purification process. These process steps are the link between the synthesis and the purification of the therapeutic products. They are critical to yield the product in its native form from the production system and require careful optimization.