Adapting virus filtration to continuous processing: Effects of product and process variability on filtration performance.

Virus filtration (VF) is an important unit operation in the manufacture of biotherapeutics that provides robust removal of potential virus contaminants. Small virus removal can be impacted by the low operating pressures and potential depressurization events that are often associated with continuous operations where increased operational flexibility for higher loading at low flux and low pressure is required. In this study, we evaluated the impact of low flux (7 LMH) and pressure interruptions on minute virus of mice (MVM) removal. We used long-term filtrations conducted to a target throughput of 1000 L/m2 with four different monoclonal antibodies on small-scale hollow fiber virus filters with a hydrophilic modified polyvinylidene fluoride membrane. These conditions are certainly challenging for any VF operation and ensuring robust viral clearance under such conditions is critical to the design and implementation of continuous VF. Planova BioEX filters effectively removed MVM at 4 log or greater when run continuously for up to 6 days. Interestingly, pressure increases associated with filter fouling over the duration of long-term filtrations were shown to be reflective of load material variability and could be remediated by implementation of an inline prefilter. Pressure interruptions had minimal impact on overall MVM logarithmic reduction value. Effective virus removal was achieved with pressure increases being largely product-specific, which demonstrates the capability of the virus filter to remove virus independent of pressure increases that are expected to occur with increased protein load.

Serial virus filtration: A case study evaluating the product-dependent impact of control strategies on process efficiency.

The production of biopharmaceutical products carries an inherent risk of contamination by adventitious viruses. Historically, these manufacturing processes have incorporated a dedicated virus filtration step to ensure product safety. However, challenging process conditions can lead to passage of small viruses to the permeate pool and an overall decrease in the desired virus logarithmic reduction value (LRV) for the process. The implementation of serial virus filtration has improved the robustness of such processes, albeit concerns about increased operating times and process complexity have limited its implementation. This work focused on optimizing a serial filtration process and identifying process control strategies to provide maximum efficiency while ensuring proper controls for process complexity. Constant TMP was identified as the optimal control strategy, which combined with the optimal filter ratio, resulted in a robust and faster virus filtration process. To demonstrate this hypothesis, data with two filters connected in series (1:1 filter ratio) are presented for a representative non-fouling molecule. Similarly, for a fouling product, the optimal setup was a combination of a filter connected in series to two filters operated in parallel (2:1 filter ratio). The optimized filter ratios bring cost- and time-savings benefits to the virus filtration step, thereby offering improved productivity. The results of risk and cost analyses performed as part of this study combined with the control strategy, offer companies a toolbox of strategies to accommodate products with different filterability profiles in their downstream processes. This work demonstrates that the safety advantages of performing filters in series can be achieved with minimal increases in time, cost, and risk.

Development of small-scale models to understand the impact of continuous downstream bioprocessing on integrated virus filtration.

We designed small-scale virus filtration models to investigate the impact of the extended process times and dynamic product streams present in continuous manufacturing. Our data show that the Planova 20N and BioEX virus filters are capable of effectively removing bacteriophage PP7 (>4 log) when run continuously for up to 4 days. Additionally, both Planova 20N and BioEX filters were able to successfully process a mock elution peak of increased protein, salt, and bacteriophage concentrations with only an increase in filtration pressure observed during the higher protein concentration peak. These experiments demonstrated that small-scale viral clearance studies can be designed to model a continuous virus filtration step with specific process parameters.

Dual regulatory functions of the thin filament revealed by replacement of the troponin I inhibitory peptide with a linker.

Striated muscles are relaxed under low Ca(2+) concentration conditions due to actions of the thin filament protein troponin. To investigate this regulatory mechanism, an 11-residue segment of cardiac troponin I previously termed the inhibitory peptide region was studied by mutagenesis. Several mutant troponin complexes were characterized in which specific effects of the inhibitory peptide region were abrogated by replacements of 4-10 residues with Gly-Ala linkers. The mutations greatly impaired two of troponin's actions under low Ca(2+) concentration conditions: inhibition of myosin subfragment 1 (S1)-thin filament MgATPase activity and cooperative suppression of myosin S1-ADP binding to thin filaments with low myosin saturation. Inhibitory peptide replacement diminished but did not abolish the Ca(2+) dependence of the ATPase rate; ATPase rates were at least 2-fold greater when Ca(2+) rather than EGTA was present. This residual regulation was highly cooperative as a function of Ca(2+) concentration, similar to the degree of cooperativity observed with WT troponin present. Other effects of the mutations included 2-fold or less increases in the apparent affinity of the thin filament regulatory Ca(2+) sites, similar decreases in the affinity of troponin for actin-tropomyosin regardless of Ca(2+), and increases in myosin S1-thin filament ATPase rates in the presence of saturating Ca(2+). The overall results indicate that cooperative myosin binding to Ca(2+)-free thin filaments depends upon the inhibitory peptide region but that a cooperatively activating effect of Ca(2+) binding does not. The findings suggest that these two processes are separable and involve different conformational changes in the thin filament.