Modeling the Residence Time Distribution of Integrated Continuous Bioprocesses.

In response to the biopharmaceutical industry advancing from traditional batch operation to continuous operation, the Food and Drug Administration (FDA) has published a draft for continuous integrated biomanufacturing. This draft outlines the most important rules for establishing continuous integration. One of these rules is a thorough understanding of mass flows in the process. A computer simulation framework is developed for modeling the residence time distribution (RTD) of integrated continuous downstream processes based on a unit-by-unit modeling approach in which unit operations are simulated one-by-one across the entire processing time, and then combined into an integrated RTD model. The framework allows for easy addition or replacement of new unit operations, as well as quick adjustment of process parameters during evaluation of the RTD model. With this RTD model, the start-up phase to reach steady state can be accelerated, the effects of process disturbances at any stage of the process can be calculated, and virtual tracking of a section of the inlet material throughout the process is possible. A hypothetical biomanufacturing process for an antibody was chosen for showcasing the RTD modeling approach.

Truly continuous low pH viral inactivation for biopharmaceutical process integration.

Continuous virus inactivation (VI) has received little attention in the efforts to realize fully continuous biomanufacturing in the future. Implementation of continuous VI must assure a specific minimum incubation time, typically 60 min. To guarantee the minimum incubation time, we implemented a packed bed continuous viral inactivation reactor (CVIR) with narrow residence time distribution (RTD) for low pH incubation. We show that the RTD does not broaden significantly over a wide range of linear flow velocities-which highlights the flexibility and robustness of the design. Prolonged exposure to acidic pH has no impact on bed stability, assuring constant RTD throughout long term operation. The suitability of the packed bed CVIR for low pH inactivation is shown with two industry-standard model viruses, that is xenotropic murine leukemia virus and pseudorabies virus. Controls at neutral pH showed no system-induced VI. At low pH, significant VI is observed, even after only 15 min. Based on the low pH inactivation kinetics, the continuous process is equivalent to traditional batch operation. This study establishes a concept for continuous low pH inactivation and, together with previous reports, highlights the versatility of the packed bed reactor for continuous VI, regardless of the inactivation method.

A narrow residence time incubation reactor for continuous virus inactivation based on packed beds.

A narrow residence time distribution (RTD) is highly desirable for continuous processes where a strict incubation time must be ensured, such as continuous virus inactivation. A narrow RTD also results in faster startup and shut down phases and limits the broadening of potential disturbances in continuous processes. A packed bed reactor with non-porous inert beads was developed to achieve narrow RTDs. The performance was defined as the ratio between the onset of the cumulative RTD and the median residence time (tx%/t50%). Laboratory-scale packed columns were used to study the influence of the column parameters on the RTD. A larger column with a void volume of 0.65 L and a length of 89 cm, packed with beads in a size range of 125 to 250 µm, achieved t0.5%/t50% >0.93 across flow rates from 0.1 to 9.8 mL/min. The RTD was significantly narrower than the RTDs of other reactor designs, such as the Coiled Flow Inverter and Jig in a Box. The pressure drop remained under 3 kPa for all tested flow rates. Fluorescent nanoparticles (30 and 200 nm) were used to mimic viruses. These two sizes showed less than 2% difference in terms of t1%/t50% and t0.01%/t50% scores. These results indicated that viruses travelled through the column at rates independent of size. This proposal of packed beds as incubation chambers for continuous virus inactivation is simple, scalable, and can be realized as single-use devices. Due to the low pressure drop, the system can be easily integrated into a fully continuous process.

Continuous Solvent/Detergent Virus Inactivation Using a Packed-Bed Reactor.

Continuous virus inactivation (VI) remains one of the missing pieces while the biopharma industry moves toward continuous manufacturing. The challenges of adapting VI to the continuous operation are two-fold: 1) achieving fluid homogeneity and 2) a narrow residence time distribution (RTD) for fluid incubation. To address these challenges, a dynamic active in-line mixer and a packed-bed continuous virus inactivation reactor (CVIR) are implemented, which act as a narrow RTD incubation chamber. The developed concept is applied using solvent/detergent (S/D) treatment for inactivation of two commonly used model viruses. The in-line mixer is characterized and enables mixing of the viscous S/D chemicals to ±1.0% of the target concentration in a small dead volume. The reactor's RTD is characterized and additional control experiments confirm that the VI is due to the S/D action and not induced by system components. The CVIR setup achieves steady state rapidly before two reactor volumes and the logarithmic reduction values of the continuous inactivation process are identical to those obtained by the traditional batch operation. The packed-bed reactor for continuous VI unites fully continuous processing with very low-pressure drop and scalability.

Multiple Single-Unit Long-Term Tracking on Organotypic Hippocampal Slices Using High-Density Microelectrode Arrays.

A novel system to cultivate and record from organotypic brain slices directly on high-density microelectrode arrays (HD-MEA) was developed. This system allows for continuous recording of electrical activity of specific individual neurons at high spatial resolution while monitoring at the same time, neuronal network activity. For the first time, the electrical activity patterns of single neurons and the corresponding neuronal network in an organotypic hippocampal slice culture were studied during several consecutive weeks at daily intervals. An unsupervised iterative spike-sorting algorithm, based on PCA and k-means clustering, was developed to assign the activities to the single units. Spike-triggered average extracellular waveforms of an action potential recorded across neighboring electrodes, termed "footprints" of single-units were generated and tracked over weeks. The developed system offers the potential to study chronic impacts of drugs or genetic modifications on individual neurons in slice preparations over extended times.

Biosimilar structural comparability assessment by NMR: from small proteins to monoclonal antibodies.

Biosimilar drug products must have a demonstrated similarity with respect to the reference product's molecules in order to ensure both the effectiveness of the drug and the patients' safety. In this paper the fusion framework of a highly sensitive NMR fingerprinting approach for conformational changes and mathematically-based biosimilarity metrics is introduced. The final goal is to translate the complex spectral information into biosimilarity scores, which are then used to estimate the degree of similarity between the biosimilar and the reference product. The proposed method was successfully applied to a small protein, i.e., filgrastim (neutropenia treatment), which is the first biosimilar approved in the United States, and a relatively large protein, i.e., monoclonal antibody rituximab (lymphoma treatment). This innovative approach introduces a new level of sensitivity to structural changes that are induced by, e.g., a small pH shift or other changes in the protein formulation.