Single Pass Tangential Flow Filtration for Process Intensification in Biomanufacturing

Andrew Zydney - Bayard D. Kunkle Chair and Professor of Chemical Engineering, The Pennsylvania State University

There is growing interest in the application of Single Pass Tangential Flow Filtration (SPTFF) systems for process intensification in biomanufacturing. In general, SPTFF refers to any membrane device that can provide a significant degree of product concentration (by at least a factor of 1.5) in a single pass through the module, without any hold tanks or recirculation loops. One of the distinct advantages of SPTFF is that the feed only passes through the pumps / valves / modules once, thereby minimizing the likelihood of product aggregation or denaturation during the filtration process. In addition, SPTFF is highly attractive for use as part of connected or continuous processing since the feed is directly concentrated in a single pass through the module.

The high degree of product concentration in SPTFF is accomplished using modules with relatively long path-lengths and/or by operating the modules at relatively low feed flow rates. Although the use of low feed flow rates may seem counterintuitive, it is important to remember that the filtrate flux during tangential flow filtration varies with the feed flow rate by an exponent less than one, i.e.,

where n ≈ 1/3 for protein ultrafiltration in hollow fiber modules and open cassettes while n ≈ ½ for spiral wound modules and cassettes with internal screens. Somewhat higher exponents are often obtained in microfiltration systems due to the enhanced transport provided by the large cells. Thus, increasing the feed flow rate will increase the filtrate flux, but the concentration factor (which is determined by the ratio of the permeate to feed flow rates) decreases with increasing feed flow rate due to the n-1 power dependence on q_feed.

Although there are a number of potential opportunities for using SPTFF for process intensification in biomanufacturing, the different applications are conveniently categorized as either post-concentration, pre-concentration, or formulation:

Post-concentration: SPTFF can be used to reduce the volume of intermediate pools by inline concentration of the clarified cell culture fluid obtained from a perfusion bioreactor or the eluted product obtained from Protein A or ion exchange chromatography columns. This enables the use of smaller pool tanks, thereby debottlenecking the downstream bioprocessing capacity. This is of particular interest in managing the operation of existing manufacturing facilities in response to changes in the upstream process, e.g., the conversion of fed-batch to perfusion processes or the improvement in productivity of the bioreactor. For example, monoclonal antibody (mAb) titers have increased by nearly 10-fold over the past decade, requiring the use of larger chromatography columns for initial product capture. The pool tanks in existing manufacturing facilities will often be too small to handle the increased elution volumes from these columns, making it difficult to fully take advantage of the increased product titer. SPTFF can eliminate these tankage constraints by reducing the volume of the elution stream, allowing manufacturers to accommodate the increased productivity without having to install larger storage tanks (which may not be practical given the limited floorspace in the manufacturing facility). Dizon-Maspat et al. (2012) have demonstrated the use of SPTFF for post-concentration of elution streams in several mAb processes, with concentration factors ranging from 2- to 10-fold. 

Pre-concentration: SPTFF can also be used for inline concentration to increase the performance of subsequent unit operations in the downstream process. For example, Host Cell Protein (HCP) removal in Anion Exchange Chromatography (AEX) is often limited by the very low concentration of HCP in the feed. The dynamic binding capacity of the AEX column can be increased by pre-concentrating the feed to shift the binding isotherm closer to saturation. Elich et al. (2019) used SPTFF to improve the overall performance of a mAb process involving Protein A, cation exchange, and then anion exchange chromatography, with the AEX performed in flow-through mode to remove trace HCP. The use of SPTFF before the final AEX step gave a 4-fold increase in mAb loading with equivalent HCP and virus removal, significantly increasing the overall productivity of the downstream process. It is also possible to use SPTFF to improve the performance of the Protein A chromatography step, with the lower feed volume leading to shorter load times in multi-cycle/multi-column systems.

Formulation: SPTFF is also attractive for providing the very high product concentrations (>200 g/L) needed for dosing of many mAb therapeutics. In addition to minimizing pump passes, SPTFF modules have smaller working volumes due to the elimination of the recirculation loop and the ability to use smaller size piping (due to the lower feed flow rates). The net result is an increase in overall product recovery, while avoiding the need to over-concentrate the product to accommodate the dilution that occurs during flushing of the system to enhance product recovery. Casey et al. (2011) demonstrated that SPTFF could be used to concentrate a 45 g/L IgG solution to ≈200 g/L, with stable operation obtained over 10 repeat operating cycles.

Buffer Exchange

Although not as widely implemented in current bioprocessing, SPTFF can also be used for buffer exchange and desalting in place of traditional batch diafiltration. In contrast to batch diafiltration, in which the diafiltration buffer is typically added at the same time permeate (containing the old buffer) is removed, buffer exchange by SPTFF occurs by first diluting the feed with the diafiltration (or formulation) buffer and then re-concentrating by SPTFF. Thus, a 2-fold reduction in salt concentration can be achieved by 1:1 dilution of the feed with deionized water followed by re-concentration to the original volume or flow rate. The low levels of buffer exchange that would typically be appropriate for in-process adjustment of pH or conductivity (e.g., to optimize the performance of an ion exchange chromatography step) can be accomplished using a single stage dilution/re-concentration. However, final formulation requires much higher levels of buffer exchange, which can only be practically achieved using some type of staged operation. Without staging, a 99.9% buffer exchange would require dilution in a 999:1 ratio, which would not only use ridiculous amounts of buffer, it would also require impractically large SPTFF devices for the re-concentration step.

Systems employing sequential (or co-current) dilution/re-concentration are the simplest to operate, with the degree of impurity removal given as:

where R is the ratio of the impurity concentration in the feed to that in the formulated product stream, α is the ratio of the flow of the dilution buff er to that of the feed, and N is the number of states. The inline diafiltration (ILDF) module developed by Pall Corporation uses six stages with α = 2.2 in each stage, yielding a system that can reduce the concentration of the penultimate buffer by approximately 1000-fold (99.9% buff er exchange). Jabra et al. (2019) demonstrated that countercurrent staging can significantly reduce the buffer requirements, while increasing the efficiency of the diafiltration, by effectively “re-using” the diafiltration buffer between the stages as shown schematically in Figure 1.

For example, the degree of impurity removal for a countercurrent staged system is given as:

Thus, the 3-stage system shown in Figure 1 can provide 99.9% buffer exchange with α = 9.6, which is about 30% less buffer than that used in the co-current staged system (with one-half the number of SPTFF modules). Jabra et al. (2019) successfully employed this 3-stage system for 24 hr of continuous operation without any degradation in membrane performance or need for cleaning/ regeneration of the module.

Continuous Processing

SPTFF can also be an enabling technology for the development of continuous biomanufacturing. A number of recent review articles have discussed the benefits of continuous operations including lower capital costs, greater flexibility in manufacturing, and the potential for improved product quality due to the shorter and more uniform residence time. SPTFF can be used in these continuous processes for both inline concentration and buffer exchange to adjust product concentration, pH, and conductivity to maintain optimal operating conditions for the individual unit operations even in response to upstream disturbances in the continuous process.

There are also exciting opportunities for using SPTFF as part of novel operations specifically designed for continuous bioprocessing. For example, Dutta et al. (2015) describe the use of SPTFF as part of a continuous countercurrent tangential chromatography system employing a chromatographic resin in the form of a flowing slurry for mAb purification. In this case, SPTFF is used for inline concentration of the resin slurry in the washing and elution steps (after dilution of the resin with the wash and elution buffers, respectively). Again, the SPTFF units are arrayed in a countercurrent staged configuration to enhance the impurity removal and product recovery while minimizing the amount of buffer required. The large pore size hollow fiber modules used in this application can provide high conversion (>70%) while maintaining high filtrate flux. A similar approach can be used for mAb purification by precipitation, with the precipitated protein dewatered and washed using countercurrent staged SPTFF modules operated below the critical filtrate flux associated with membrane fouling (Li et al., 2019).

SPTFF Module Design

In contrast to batch TFF systems in which one can adjust the processing time to achieve the desired degree of final concentration, SPTFF modules must be specifically designed to provide the required concentration factor in a single pass. This can be accomplished using multiple TFF cassettes in a series arrangement to obtain a long path-length module, or multiple cassettes can be used in parallel to increase the surface area and thus increase the conversion. Jabra et al. (2022) have examined the performance characteristics of SPTFF modules as part of a major research project supported through the National Science Foundation Industry-University Cooperative Research Center (NSF IUCRC) in Membrane Science, Engineering, and Technology (the MAST Center). Experimental studies were performed using a mAb provided by Biogen, one of the Center’s Industry Advisory Board (IAB) members, with SPTFF modules having different geometries provided by Pall Corporation and MilliporeSigma (both members of the IAB). Parallel arrangements of modules could be used to achieve the desired concentration factor with low feed-side pressure drops but at the cost of greater membrane surface area. The membrane area could be significantly reduced by using a series arrangement to create a single long path-length channel, but these modules had very high pressure drops. Jabra et al. (2022) also examined the use of staged configurations to achieve the optimal combination of concentration factor and pressure drop. For example, the Cadence SPTFF module developed by Pall uses an internally staged configuration in which the number of parallel cassettes is decreased as one moves along the length of the module to increase the feed velocity (and thus the shear rate) as permeate is removed. Figure 2 shows a 3-2-1-1 configuration in which the feed is initially split among 3 parallel modules followed by a region with 2 parallel modules and then the final two regions with only a single module, giving a long path-length but somewhat lower pressure drop.

The opportunities for using SPTFF technology are likely to grow as biopharmaceutical companies continue to pursue the development of connected unit operations and fully integrated continuous bioprocessing for the manufacture of high value biotherapeutics. This will include the production of new monoclonal antibody products, particularly ones for large demand applications, as well as development of SPTFF technology for the production of vaccines and gene therapies. SPTFF not only has the potential to reduce manufacturing costs through process intensification, it can also simultaneously improve product quality by reducing the number of pump passes and processing time.

References

1. Casey, C., Gallos, T., Alekseev, Y., Ayturk, E., Pearl, S. (2011) Protein concentration with single-pass tangential flow filtration (SPTFF), Journal of Membrane Science, 384: 82–88. https://doi.org/10.1016/j.memsci.2011.09.004.
2. Dizon-Maspat, J., Bourret, J., D’Agostini, A., Li, F. (2012) Single pass tangential flow filtration to debottleneck downstream processing for therapeutic antibody production, Biotechnology and Bioengineering, 109: 962–970. https://doi.org/10.1002/bit.24377.
3. Dutta, A. K., Tran, T., Napadensky, B., Teella, A., Brookhart, G., Ropp, P. A., Zhang, A. W., Tustian, A. D., Zydney, A. L., & Shinkazh, O. (2015) Purification of monoclonal antibodies from clarified cell culture fluid using Protein A capture continuous countercurrent tangential chromatography. Journal of Biotechnology, 213: 54–64. https://doi. org/10.1016/j.jbiotec.2015.02.026
4. Elich, T., Goodrich, E., Lutz, H., Mehta, U. (2019) Investigating the combination of single-pass tangential flow filtration and anion exchange chromatography for intensified mAb polishing, Biotechnology Progress, 35: e2862. https://doi.org/10.1002/btpr.2862.
5. Jabra, M. G., Yehl, C. J., Zydney, A. L. (2019) Multistage continuous countercurrent diafiltration for formulation of monoclonal antibodies. Biotechnology Progress, 35(4): e2810. https://doi.org/10.1002/btpr.281
6. Jabra, M.G., Zydney, A.L. (2022) Design and optimization of Single Pass Tangential Flow Filtration for inline concentration of monoclonal antibodies, Journal of Membrane Science, 643: 120047. https://doi.org/10.1016/j.memsci.2021.120047
7. Li, Z., Gu, Q., Coff man, J. L., Przybycien, T., & Zydney, A. L. (2019) Continuous precipitation for monoclonal antibody capture using countercurrent washing by microfi ltration. Biotechnology Progress, 35(6): e2886. https://doi.org/10.1002/btpr.2886
8. Zydney, A.L. (2021) New developments in membranes for bioprocessing – A review, Journal of Membrane Science, 620: 118804. https://doi.org/10.1016/j.memsci.2020.118804 

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