Protein concentration with single-pass tangential flow filtration (SPTFF)

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Abstract

This paper presents a novel Cadence™ single-pass tangential flow filtration (SPTFF) process for protein concentration, operating in a continuous mode. The main advantages of the SPTFF technology have been highlighted in a benchmark comparison test versus conventional TFF. SPTFF modules consist of internally staged T-series cassettes for creating longer flow paths that result in significantly higher conversion in one pass. This eliminates the need for the conventional TFF recirculation loop. SPTFF utilizes flow ratio control to achieve target concentration factors and final product concentrations instantaneously. Bovine IgG solutions in PBS (5–45 g/L) were processed to achieve a concentration factor range of 3–25×, which resulted in permeate fluxes in the range of 10–70 L/m2/h over a feed-side pressure drop range of 25–65 psid. Ten cycles of high concentration, product recovery and cleaning were successfully completed with the same SPTFF module to demonstrate experimental reproducibility and process stability. A ∼45 g/L IgG feed solution was concentrated to 190–225 g/L during all cycles resulting in high product recoveries in the range of 95.7–98.9%.

Highlights

► Continuous processing, high concn factors and high conversions in single-pass. ► No recirc. loop, min. aggregation, no mixing and min. shear exposure. ► Low-cost systems, lower system holdup and flush volumes and high recoveries. ► In-process volume and inline salt reduction, high-concn formulations. ► Viable option for the processing of fragile biomolecules.

Introduction

As a key bioprocess unit operation, ultrafiltration is mainly utilized for the concentration, buffer exchange, and purification of proteins, and often used multiple times during downstream processing [1]. Therefore, the performance of the ultrafiltration unit operation has a direct impact on the overall process yield.

The deterioration of the permeate flux, which is a critical determinant of the performance of pressure-driven membrane processes, generally takes place due to the transient build-up of retained solutes such as ions, macro-molecules and suspended colloids and/or the presence of high solute concentrations at the bulk–membrane interface, a phenomenon also known as concentration polarization [2]. Tangential flow filtration (TFF) reduces the adverse effects of the concentration polarization layer and improves the overall separation performance, and therefore has been widely accepted due to its inherent advantages over dead-ended or direct flow filtration (DFF) [3].

In TFF, while the trans-membrane pressure (TMP) drives fluid through the membrane, the cross-flow velocity, defined as the rate of the fluid flow through the feed channel and across the membrane, provides the force that sweeps away molecules from the surface. Therefore, polarized solutes are swept away from the membrane surface, which increases back-diffusion and minimizes the decline in performance due to membrane fouling and reduced filtrate flow [2]. In comparison to DFF, reduced membrane fouling, filtrate flow stability and the reusability of TFF modules not only enhance the lifetime and throughput of the TFF membranes but also lower the costs. TFF processing has been a ubiquitous unit operation for the upstream recovery and downstream purification of biomolecules in numerous bioprocess applications [4]. A wide range of TFF applications include but are not limited to the concentration and desalting of proteins [5], [6], [7], peptides [8] and nucleic acids (i.e., DNA/RNA/oligonucleotides), recovery and purification of antibodies or recombinant proteins from cell culture media, plasmid DNA from cell lysates or chromosomal DNA from whole blood [9], [10], [11], fractionation of dilute protein mixtures [12], clarification of cell lysates or tissue homogenates and depyrogenation (endotoxin removal) from water, buffers, and media solutions [13], cell harvesting and recovery or removal of viruses [14], [15], [16].

The majority of TFF processes are operated in batch-mode, and a drawback of the conventional TFF process is the low conversion per pass, which thereby requires a recirculation loop, a large feed pump and a relatively large feed tank [17]. In addition, mixing and aggregation can be problematic and foaming may further reduce the product yields and quality. Due to the large recirculation tanks, conventional TFF systems have large minimum working volumes, which limits the maximum achievable concentration factor (CF) for a given batch and also complicates the product recovery. Conventional TFF would require intermediate holding tanks if used for in-process volume reduction, necessitating added space and clean-up. Moreover, the feed material passes through the pump multiple times, which can be damaging to shear-sensitive and fragile biomolecules [18].

Most recently, the single-pass tangential flow filtration (SPTFF) process, a novel approach to concentration with TFF, has been developed [19]. The SPTFF process is continuous and employs a stepwise change in flow path through multiple TFF cassettes, concentrating the feed material in one pass through the module. The stepwise decrease in the number of feed flow channels allows for optimization of feed flow velocities and performance, as permeate is removed from each stage. The feed is exposed to only a single pump pass, resulting in lower shear exposure than in conventional TFF. SPTFF also leads to lower flow rates, so smaller, less expensive pumps and tubing can be utilized. Additional benefits over conventional TFF include lower system hold-up volumes, higher recoveries, and lower flush volume requirements [19]. Elimination of the conventional recirculation loop decreases aggregation problems and requires no mixing, and also allows the SPTFF step to be coupled with other downstream process steps such as virus removal and chromatography for higher overall throughput and reduced costs [18]. Because the feed material concentrates as it moves through the SPTFF module, very high concentrations factors (CF) can be achieved in one pass. High concentrations are especially desirable for drug formulations for subcutaneous administration [20], [21]. However, the main challenge yet to overcome while processing highly concentrated solutions is to establish a balance between process stability and the processing fluid characteristics that are normally accompanied by lower fluxes, higher viscosities, and a risk of overconcentration [21]. Indeed, for conventional TFF, concentration factors are limited by the minimum working volume of the system. This limits the maximum concentration that can be reached without having a second system to reach the final concentration.

The main objective of the current study was to understand the fundamental operating principles of the novel SPTTF technology and to elucidate the main advantages with respect to conventional TFF unit operation. For this purpose, benchmarking experiments providing a thorough understanding of single-pass, continuous mode operation and the flow ratio control (FRC) principle, were carried out utilizing IgG solutions at varying feed conditions. Then, the impact of flow path on the process capability was studied in a single SPTFF module covering a CF range of 3–25×. Finally, cycle testing was conducted to demonstrate the reproducibility and stability of a SPTFF module at high IgG concentrations.

Section snippets

Materials and methods

Regenerated cellulose (delta) membranes with a molecular weight cutoff (MWCO) of 10 kDa were used for all studies. All membranes were supplied from Pall Corporation (Northborough, MA) in a T-Series T01 Centramate™ format with an area of 0.0093 m2 (0.1 ft2). Individual T01 cassettes were stacked in parallel in the conventional setup, and the same cassettes were used to construct the SPTFF modules with internally staged flow paths. The SPTFF modules with flow path configurations used in this study

Conventional vs. single-pass tangential flow filtration

The conventional TFF unit operation typically requires the optimization of key operating parameters based on the specifications and the limitations of the process. Fig. 3 shows the flux excursion profiles of a conventional TFF module of 0.12 m2 total area, which consisted of thirteen (13) T01 cassettes stacked in parallel. A 5.2 g/L IgG solution was processed at three (3) retentate CFFs of 1.08, 3.23 and 5.38 L/min/m2 (0.1, 0.3 and 0.5 L/min/ft2). While the increase in filtrate flux as a function

Conclusions

A novel single-pass TFF device that operates in continuous mode and eliminates the conventional TFF recirculation loop has been presented. A ∼5 g/L IgG solution was concentrated 6–7 fold with both conventional and single-pass TFF systems to directly compare the two technologies. With SPTFF, the feed and retentate flow rates were set through flow ratio control, attaining the desired concentration factor and final formulation instantly, and minimizing the number of pump passes to only one.

Acknowledgements

The authors gratefully acknowledge the contributions and support from Leon Mir and Gastón de los Reyes of SPF Innovations LLC (Somerville, MA), during the development of SPTFF technology. We also would like to thank Thomas Scholz, Cheryl Sayer, the TFF Product Development team and all the colleagues at Pall Corporation for their continuing support. Cadence™ and Centramate™ are trademarks of Pall Corporation.

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