Pilot scale implementation of a single-use, high intensity, integrated process system

Pfizer and Boehringer Ingelheim are developing pilot scale systems with entirely single-use flow paths that are fully integrated end-to-end under a single control system from bioreactor through downstream processing. These prototype systems are designed to run continuous processing from the bioreactor to downstream, and periodic processing to the end of the downstream system. This presentation shows the evolution of the systems including some novel single use technologies, details of some high-intensity run results, and offers future single use improvement ideas

GMP design of a single-use integrated continuous bio manufacturing system

This presentation will show design work by Pfizer and Boehringer Ingelheim on a single-use integrated continuous system for GMP Biomanufacturing including consideration of scale, facility-fit, automation, single-use devices, hardware, and process control and monitoring. The scale of the system needs to be appropriate for the expected quantities of drug substance needed and needs to fit within the constraints of the GMP facility, including physical size and interaction with other facility systems. The system automation needs to control and monitor the entire process, since the upstream and downstream are integrated and the downstream operates semi-continuously. This presentation will discuss the challenges of designing an automation scheme capable of controlling an integrated upstream and downstream process along with the unique features that proved enabling to the integrated system. The single use devices and instruments in the system, including those made with additive manufacturing, are the process contact surfaces, so they need to be compatible with all the process fluids, perform consistently over process cycling and be constructed with sanitary design appropriate for GMP biomanufacturing. The system hardware provides the interface between the automation and the single-use devices controlling operations and the single-use instruments monitoring in-line process data. The design also needs to consider on-line instrument needs and off-line sampling analysis for a continuous flowing process stream

iSKID: From integrated pilot scale runs to GMP implementation approach

One of the most compelling business reasons for integrated processing is the ability to de-risk capital investment due to a significantly more productive process that takes less space and fewer campaigns to generate clinical and commercial material. Boehringer Ingelheim and Pfizer developed the iSKID, a fully integrated and automated system that hydraulically links the perfusion bioreactor with several downstream unit operations (2xProtein A columns, continuous viral inactivation, AEX in flow through mode, and SPTFF). The Protein A elution cycles are discrete and separated by \u3e2hrs, allowing the ability to discard cycles that do not meet process specifications. The discreteness between product cycles and hydraulic linkage enables the sanitization between cycles for a robust bioburden control strategy. Each cycle is captured in a single use mixer (SUM), where the product is pooled in stable conditions until viral filtration, ultrafiltration/diafiltration and final filtration are performed in batch mode. Identical iSKID prototypes at 100L scale were used at three different sites to generate product quality, process, and bioburden data from three different molecules. The data has been used to understand implementation gaps in GMP facilities and process platforms (CMC1/CMC2). In addition, the team identified specific items to present to the FDA’s Emerging Technology Team (ETT). These items include our strategies for batch definition, microbial control, and process control. In this talk, we will use the data generated from the consistency runs to elaborate on the robustness of the process and touch upon the strategies to be presented to the ETT

Utilizing high-throughput experimentation to enhance specific productivity of an E.coli T7 expression system by phosphate limitation

<p>Abstract</p> <p>Background</p> <p>The specific productivity of cultivation processes can be optimized, amongst others, by using genetic engineering of strains, choice of suitable host/vector systems or process optimization (e.g. choosing the right induction time). A further possibility is to reduce biomass buildup in favor of an enhanced product formation, e.g. by limiting secondary substrates in the medium, such as phosphate. However, with conventional techniques (e.g. small scale cultivations in shake flasks), it is very tedious to establish optimal conditions for cell growth and protein expression, as the start of protein expression (induction time) and the degree of phosphate limitation have to be determined in numerous concerted, manually conducted experiments.</p> <p>Results</p> <p>We investigated the effect of different induction times and a concurrent phosphate limitation on the specific productivity of the T7 expression system <it>E.coli </it>BL21(DE3) pRhotHi-2-EcFbFP, which produces the model fluorescence protein EcFbFP upon induction. Therefore, specific online-monitoring tools for small scale cultivations (RAMOS, BioLector) as well as a novel cultivation platform (Robo-Lector) were used for rapid process optimization. The RAMOS system monitored the oxygen transfer rate in shake flasks, whereas the BioLector device allowed to monitor microbial growth and the production of EcFbFP in microtiter plates. The Robo-Lector is a combination of a BioLector and a pipetting robot and can conduct high-throughput experiments fully automated. By using these tools, it was possible to determine the optimal induction time and to increase the specific productivity for EcFbFP from 22% (for unlimited conditions) to 31% of total protein content of the <it>E.coli </it>cells via a phosphate limitation.</p> <p>Conclusions</p> <p>The results revealed that a phosphate limitation at the right induction time was suitable to redirect the available cellular resources during cultivation to protein expression rather than in biomass production. To our knowledge, such an effect was shown for the first time for an IPTG-inducible expression system. Finally, this finding and the utilization of the introduced high-throughput experimentation approach could help to find new targets to further enhance the production capacity of recombinant <it>E.coli</it>-strains.</p

The Eu:CROPIS Mass Property Campaign: Trimming a spin-stabilized compact satellite for a long term artificial gravity experiment

Eu:CROPIS (Euglena Combined Regenerative Organic Food Production In Space) is the first mission of DLR's Compact Satellite program. The launch of Eu:CROPIS took place on December 3rd in 2018 on-board the Falcon 9 SSO-A mission. The satellite’s primary payload Eu:CROPIS features a biological experiment in the context of closed loop coupled life support systems. The Eu:CROPIS satellite mission uses spin stabilization along its z-axis to provide defined acceleration levels for the primary and secondary payloads to simulate either a Moon or Mars gravity environment. For the payload performance it is vital to achieve a minimum deviation between spacecraft z-axis and the major moment of inertia (MoI) axis to minimize the offset of the envisaged acceleration levels. Specific moment of inertia ratios between the spin- and minor axes had to be maintained to allow the attitude control system to keep the satellite at a stable rotation despite environmental disturbances. This paper presents the adaptive and flexible trimming strategy applied during the flight model production, as well as the mass properties measurement acceptance campaign and the respective results

The Eu:CROPIS Mass Property Campaign: Trimming a spin-stabilized compact satellite for a long term artificial gravity experiment

Eu:CROPIS (Euglena Combined Regenerative Organic Food Production In Space) is the first mission of DLR's Compact Satellite program. The launch of Eu:CROPIS took place on December 3rd in 2018 on-board the Falcon 9 SSO-A mission. The satellite’s primary payload Eu:CROPIS features a biological experiment in the context of closed loop coupled life support systems. The Eu:CROPIS satellite mission uses spin stabilization along its z-axis to provide defined acceleration levels for the primary and secondary payloads to simulate either a Moon or Mars gravity environment. For the payload performance it is vital to achieve a minimum deviation between spacecraft z-axis and the major moment of inertia (MoI) axis to minimize the offset of the envisaged acceleration levels. Specific moment of inertia ratios between the spin- and minor axes had to be maintained to allow the attitude control system to keep the satellite at a stable rotation despite environmental disturbances. This paper presents the adaptive and flexible trimming strategy applied during the flight model production, as well as the mass properties measurement acceptance campaign and the respective results

Determination of Radiographic Healing: An Assessment of Consistency Using RUST and Modified RUST in Metadiaphyseal Fractures

Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. Objective: To determine the reliability of the Radiographic Union Scale for Tibia (RUST) score and a new modified RUST score in quantifying healing and to define a value for radiographic union in a large series of metadiaphyseal fractures treated with plates or intramedullary nails. Design: Healing was evaluated using 2 methods: (1) evaluation of interrater agreement in a series of radiographs and (2) analysis of prospectively gathered data from 2 previous large multicenter trials to define thresholds for radiographic union. Intervention: Part 1: 12 orthopedic trauma surgeons evaluated a series of radiographs of 27 distal femur fractures treated with either plate or retrograde nail fixation at various stages of healing in random order using a modified RUST score. For each radiographic set, the reviewer indicated if the fracture was radiographically healed. Part 2: The radiographic results of 2 multicenter randomized trials comparing plate versus nail fixation of 81 distal femur and 46 proximal tibia fractures were reviewed. Orthopaedic surgeons at 24 trauma centers scored radiographs at 3, 6, and 12 months postoperatively using the modified RUST score above. Additionally, investigators indicated if the fracture was healed or not healed. Main Outcome Measures: The intraclass correlation coefficient (ICC) with 95% confidence intervals was determined for each cortex, the standard and modified RUST score, and the assignment of union for part 1 data. The RUST and modified RUST that defined union were determined for both parts of the study. Results: ICC: The modified RUST score demonstrated slightly higher ICCs than the standard RUST (0.68 vs. 0.63). Nails had substantial agreement, whereas plates had moderate agreement using both modified and standard RUST (0.74 and 0.67 vs. 0.59 and 0.53). Union: The average standard and modified RUST at union among all fractures was 8.5 and 11.4. Nails had higher standard and modified RUST scores than plates at union. The ICC for union was 0.53 (nails: 0.58; plates: 0.51), which indicates moderate agreement. However, the majority of reviewers assigned union for a standard RUST of 9 and a modified RUST of 11, and \u3e90% considered a score of 10 on the RUST and 13 on the modified RUST united. Conclusions: The ICC for the modified RUST is slightly higher than the standard RUST in metadiaphyseal fractures and had substantial agreement. The ICC for the assessment of union was moderate agreement; however, definite union would be 10 and 13 with over 90% of reviewers assigning union. These are the first data-driven estimates of radiographic union for these scores.