Protein Processing

Sample concentration and buffer exchange are integral to any preparative scale protein production, whether for structural biology or general biochemistry needs. These steps are required to ensure specific chromatographic requirements in between the purification steps. Even after purification, the protein must be formulated in a specific buffer at appropriate concentrations to ensure its structural integrity and effectiveness for downstream applications. Similarly, buffer exchange may also be required for in vitro refolding of the solubilized inclusion bodies by controlled denaturant removal.

Given the importance of these steps, factors such as gentleness, time efficiency, scalability, and cost-effectiveness must be taken into account while selecting a method for protein processing. Tables 1 and 2 outline the typical methods employed for protein concentration and buffer exchange, respectively:

Considerations	Column Chromatography	Lyophilization	Evaporation	Absorption	Ultrafiltration Dead-end TFF
Gentleness	✔			✔	✔ ✔
Scalability	✔	✔	✔	✔	✔
Time Efficiency					✔
Cost Efficiency		✔	✔		✔ ✔
Automation					✔

Table 1. Methods for protein concentration

Ultrafiltration addresses all the concerns for protein concentration as well as buffer exchange compared to conventional methods. However, dead-end ultrafiltration creates a concentration gradient at the membrane as the sample is not recirculated. This slows down the filtration process and may result in material loss due to aggregation. The only way to monitor the concentration progress or to mix the sample, is by periodic interruption of the centrifugation, making it a hands-on method and adding further to the processing time. Lastly, the dead-end centrifugal devices lack scalability and can process only limited volumes.

These concerns are effectively addressed by tangential flow filtration (TFF), an alternative form of ultrafiltration that involves constant sample recirculation, thus avoiding the filter cake formation and ensuring high filtration rates. TFF also offers scalability and advantage of simultaneous buffer exchange and sample concentration. However, traditional TFF systems have a large footprint with hold-up volumes in tens to hundreds of milliliters, making them unsuitable for lab scale applications.

Considerations	Column Chromatography	Dilution	Equilibrium Dialysis	Ultrafiltration Dead-end TFF
Gentleness	✔	✔	✔	✔ ✔
Scalability		✔	✔	✔
Time Efficiency				✔
Cost Efficiency				✔ ✔
Automation				✔

Table 2. Methods for buffer exchange

Protein Processing Using the µPulse - TFF System

The Formulatrix µPulse ^® is a fully automated and miniaturized TFF system, explicitly designed for sample concentration and diafiltration at lab scale. The entire fluid path is miniaturized on the filter chip by combining microfluidic pumping technology with TFF. This has reduced the hold-up volume to just 0.65 mL, ensuring maximum sample recovery.

The µPulse filter chips are currently available with modified polyethersulfone (mPES) and regenerated cellulose (RC) membranes that exhibit low fouling characteristics and are compatible with a variety of sample types. The µPulse offers customization for the operating pressures to make the process gentle, ensuring the structural integrity of proteins and other biomolecules. Furthermore, the µPulse processes samples 4x faster compared to dead-end centrifugal filters, and in a walk-away approach.

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High-Throughput Protein Processing with the aµtoPulse - TFF System

The aµtoPulse^® is a fully automated, high-throughput TFF system with the world’s lowest hold-up volume of just 250 µL. It processes up to 54 samples per run, with up to 4 samples in parallel. Designed for flexibility, it handles starting volumes between 0.5 mL to 100 mL and can concentrate samples down to 250 µL with ±25 µL precision. The system supports up to four external buffer inputs or on-deck conical tubes, enabling automated multi-buffer diafiltration.

The advanced chip design with dual pumps delivers permeate flow rates up to 1.7x faster than the µPulse while minimizing shear, particularly important for delicate proteins and complexes. The chips are available with mPES (5–300 kDa) and RC (5–100 kDa) membranes, ensuring compatibility with a wide range of samples.

Each station offers independent regulation and monitoring of transmembrane pressure (0–32 psi), giving users full control over process gentleness and efficiency. The intuitive, browser-based software enables remote protocol setup, monitoring, and control, with secure data management compliant with 21 CFR Part 11 for GMP environments.

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Webinars

From Harvesting to Formulation: Accelerate the Processing of Proteins and Virus Like particles with the µPulse - TFF system

Learn a fast, gentle, and automated approach for harvesting extracellular VLPs as well as refolding, and formulating various proteins using the µPulse.

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Purification of Antibody Drug Conjugates with the
µPulse - TFF System

Experience the single-step and scalable purification of ADCs and other macromolecules modified with small molecules using the µPulse.

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Application Notes

Fast Formulation of a Purified Recombinant L-asparaginase Using the µPulse - TFF System

Uncover the ease of use, scalability, and reusability of µPulse for lab scale formulation of recombinant L-asparaginase and other biomolecules.

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Fast Lab Scale Protein Refolding by Filter Dialysis on the µPulse - TFF System

Learn about the time efficiency, cost effectiveness and gentleness of µPulse for refolding denatured proteins compared to equilibrium dialysis.

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Publications

Citations: 4

A four-stage process that identified a hydroxypropyl beta cyclodextrin and polysorbate containing formulation that eliminated aggregation of recombinant human NELL-1 after exposure to extrinsic stresses

Xin et al., 2025 |AAPS Open |Link

Abstract Background Recombinant human NELL- rhNELL- is a potent osteogenic protein with therapeutic potential in regenerative medicine A stable formulation is essential to prevent aggregation during production filling storage and clinical use Methodology A four-stage rational formulation strategy was used identify intrinsic aggregation risks of rhNELL- screen polysorbate- and cyclodextrin-based ...More |

Dual Targeting for Enhanced Tumor Immunity: Conditionally Active CD28xVISTA Bispecific Antibodies Promote Myeloid-Driven T-Cell Activation

Thisted et al., 2025 |bioRxiv |Link

Reinvigoration of tumor-reactive T-cells using co-stimulatory bispecific antibodies bsAbs targeting CD or CD is emerging as a promising therapeutic strategy Conditional tumor-specific recruitment can offer a necessary layer of control and specificity We developed pH-selective CD xVISTA bsAbs to act specifically within the acidic tumor microenvironment TME aiming for enhanced ...More |

Reinvigoration of tumor-reactive T-cells using co-stimulatory bispecific antibodies (bsAbs) targeting CD28 or CD137 is emerging as a promising therapeutic strategy. Conditional, tumor-specific recruitment can offer a necessary layer of control and specificity. We developed pH-selective CD28xVISTA bsAbs to act specifically within the acidic tumor microenvironment (TME), aiming for enhanced T-cell-mediated cancer cell killing while minimizing systemic T-cell activation and Cytokine Release Syndrome (CRS) risk. CD28 agonism by CD28xVISTA bsAbs relies on pH-selective engagement of VISTA, a protein robustly expressed on myeloid cells highly prevalent in most solid tumors. This modality avoids engagement of tumor-associated antigens (TAAs) with the potential to provide highly tumor specific activity with minimal on-target/off-tumor side effects.

We report the identification of a lead candidate with pH-dependent simultaneous engagement of both targets, and VISTA-dependent CD28 signaling in a reporter cell line. CD28xVISTA avidly bound VISTA-positive cells, and co-stimulation was shown in vitro by its ability to activate and expand T-cells and enhance T-cell mediated cancer cell killing in co-cultures of human PBMCs and cancer cells in the presence of a TAA-targeted anti-CD3 T-cell engager. Interestingly, our findings support both signaling in cis (between T-cell and cell displaying peptide-MHC complex) and in trans with stimulation occurring through CD28 clustering outside of the immune synapse. Our lead candidate displayed efficient tumor growth inhibition of human VISTA-expressing MC38 cells in a humanized CD28 syngeneic mouse model in combination with PD-1 blockade. Importantly, our CD28xVISTA bsAb showed no signs of superagonistic properties in several in vitro assays geared towards revealing induction of CRS. Our data supports clinical development in combination with anti-PD-1 or any TAA-targeted anti-CD3 T-cell engagers developed for solid tumors. Less |

Rapid Development of High Concentration Protein Formulation Driven by High-Throughput Technologies

Xin et al., 2025 |Pharmaceutical Research |Link

Background High concentration protein formulation HCPF development needs to balance protein stability attributes such as conformational colloidal stability chemical stability and solution properties such as viscosity and osmolality Methodology A three-phase design is established in this work In Phase conformational and colloidal stability are measured by -well-based high-throughput HT biophysical ...More |

Structural and functional analyses of Pcal_0917, an α-glucosidase from hyperthermophilic archaeon Pyrobaculum calidifontis

Muhammad et al., 2023 |International Journal of Biological Macromolecules |Link

Genome analysis of Pyrobaculum calidifontis revealed the presence of -glucosidase Pcal gene Structural analysis affirmed the presence of signature sequences of Type II -glucosidases in Pcal We have heterologously expressed the gene and produced recombinant Pcal in Escherichia coli Biochemical characteristics of the recombinant enzyme resembled to that of Type ...More |

FAQs

Comparison to Other Methods & Workflow Integration

How does tangential flow filtration (TFF) compare to centrifugation for protein purification?
TFF is superior to centrifugation for most protein purification workflows due to its higher efficiency, scalability, and automation, while centrifugation remains useful for initial crude clarification. TFF continuously recirculates the sample across the membrane, maintaining stable flux and uniform concentration. This results in higher recovery, better reproducibility, and improved protein stability, especially for shear- or aggregation-sensitive proteins. In addition, automated TFF systems such as the µPulse and aµtoPulse enable controlled transmembrane pressure (TMP), faster processing, and integrated buffer exchange in a single run—saving time and reducing hands-on handling compared to repeated centrifugation steps.

How do I integrate TFF into an existing chromatography workflow?
Tangential flow filtration (TFF) can be integrated into an existing chromatography workflow as a complementary step for concentration and buffer exchange. Typically, after initial capture or intermediate purification using chromatography (e.g., Protein A, ion exchange, or affinity columns), the eluate can be processed via TFF to concentrate the product and exchange it into the desired formulation buffer before polishing or final formulation steps. Automated systems like µPulse and aµtoPulse simplify this integration by providing controlled transmembrane pressure, low hold-up volumes, and reproducible recovery, ensuring minimal product loss and consistent quality. Incorporating TFF in this way reduces hands-on time, increases throughput, and allows seamless scale-up from lab to pilot or GMP production.

What are the typical processing times for protein concentration and buffer exchange using TFF compared to dialysis?
TFF is significantly faster than dialysis for protein buffer exchange. While traditional dialysis can take several hours to overnight due to passive diffusion, automated TFF systems like µPulse and aµtoPulse can process small- to medium-volume protein samples in minutes to under an hour, depending on sample volume and membrane MWCO. This speed is achieved through active tangential flow, controlled transmembrane pressure, and continuous permeate removal, which not only shortens processing time but also maintains high recovery and protein integrity.

Recovery, Yield, and Performance Optimization

What recovery rates can I expect in TFF?
You can typically expect high recovery rates in TFF, often above 95%, and up to ~100% for small-volume workflows, depending on the system and membrane selection. This high recovery is achieved because tangential flow minimizes membrane fouling and aggregation while maintaining stable transmembrane pressure, which is especially important for sensitive proteins, nucleic acids, and viral vectors. Automated small-scale TFF systems such as the µPulse and aµtoPulse further improve recovery by using very low hold-up volumes (650 µL for the µPulse and 250 µL for the aµtoPulse), reducing product loss in tubing and fluid paths and ensuring that nearly all of the processed material can be recovered at the end of the run.

Best TFF configuration for high-concentration protein solutions?
The best TFF configuration for high-concentration protein solutions is tangential flow filtration operated with low, well-controlled transmembrane pressure (TMP) and high crossflow shear. This setup keeps proteins sweeping along the membrane surface instead of compacting into a dense gel layer, which helps maintain flux and prevent aggregation. Automated systems like the µPulse and aµtoPulse are particularly effective because they provide precise electronic TMP control and stable recirculation, allowing proteins to be concentrated to high levels with minimal fouling and >90–95% recovery. This matters because controlled shear and TMP preserve protein stability, improve reproducibility, and reduce product loss compared to dead-end filtration or uncontrolled centrifugation.

How does recirculation rate affect protein recovery in TFF?
The recirculation rate directly affects protein recovery in TFF by balancing shear, fouling control, and protein stability. Higher recirculation rates increase shear at the membrane surface, which reduces concentration polarization and fouling, helping maintain flux and improve recovery. However, excessive recirculation can expose proteins to unnecessary shear stress, increasing the risk of denaturation or aggregation. Automated systems such as the µPulse and aµtoPulse optimize this balance by precisely controlling flow and transmembrane pressure, ensuring sufficient recirculation to keep the membrane clean while preserving protein integrity. This matters because properly tuned recirculation maximizes recovery (typically >90–95%) while maintaining consistent product quality.

Membrane & System Selection

What MWCO membrane should I select for different protein sizes and molecular weights?
When selecting a membrane MWCO for proteins, choose a cutoff that is approximately 3–5 times smaller than the molecular weight of the target protein. This ensures the protein is fully retained while allowing smaller impurities, salts, or solvents to pass through. Automated TFF systems like µPulse and aµtoPulse support a range of 5–300 kDa MWCO membranes in RC or mPES, allowing flexibility to optimize recovery, minimize non-specific binding, and achieve efficient diafiltration or concentration for diverse protein workflows.

How scalable is protein processing with TFF from lab scale (µPulse) to high-throughput or pilot-scale (aµtoPulse)?
Protein processing with TFF is highly scalable from lab to pilot scale. Systems like the µPulse handle small-volume samples (1–100 mL) with low hold-up (650 µL), making them well suited for research and development, while the aµtoPulse extends this capability to high-throughput or pilot-scale workflows, processing up to 54 samples per run with independent pressure control and a minimum hold-up volume of 250 µL. Both systems maintain controlled transmembrane pressure, gentle tangential flow, and reproducible recovery, allowing process parameters optimized at the lab scale to be reliably transferred to larger, automated setups without compromising protein integrity or yield.

Protein Stability, Sensitive Targets & Specialized Applications

How does TFF help maintain protein stability and prevent aggregation during concentration?
TFF helps maintain protein stability and prevent aggregation by minimizing shear and concentration polarization through tangential flow across the membrane rather than dead-end filtration. Controlled transmembrane pressure (TMP) ensures proteins are gently recirculated, reducing the risk of denaturation or aggregation. Low hold-up volume systems like µPulse and aµtoPulse further reduce product loss and dead zones where aggregation could occur. Together, these features provide consistent, reproducible concentration and diafiltration while preserving the structural and functional integrity of sensitive proteins.

Can TFF systems handle sensitive proteins such as membrane proteins or enzymes without loss of activity?
Yes, TFF systems like µPulse and aµtoPulse can handle sensitive proteins, including membrane proteins and enzymes, without loss of activity. Their gentle tangential flow, controlled transmembrane pressure, and low hold-up volume minimize shear stress and aggregation, preserving protein structure and function. This allows efficient concentration and buffer exchange while maintaining high recovery, making TFF suitable for even highly labile or aggregation-prone proteins.

Can TFF be used for protein refolding workflows, such as controlled removal of denaturants?
Yes, TFF can be effectively used for protein refolding by controlled removal of denaturants. By performing diafiltration with a suitable MWCO membrane, TFF gradually removes denaturants while maintaining the protein in solution, allowing proper refolding. Automated systems like µPulse and aµtoPulse provide precise transmembrane pressure, low hold-up volumes, and gentle tangential flow, minimizing aggregation and maximizing recovery. This controlled, reproducible process enables efficient refolding of sensitive proteins while preserving structural and functional integrity.

Buffer Exchange, Diafiltration & Formulation

Can TFF be used for buffer exchange in mAbs production?
Yes, tangential flow filtration (TFF) is widely used for buffer exchange in monoclonal antibody (mAb) production. TFF enables efficient diafiltration by continuously retaining the mAb while removing salts, solvents, and small molecules, allowing rapid exchange into the desired formulation buffer. Compared to dialysis or spin columns, TFF is faster, scalable, and more reproducible, while maintaining high recovery and preserving antibody integrity through controlled shear and transmembrane pressure. This makes TFF a standard and reliable approach for buffer exchange during mAb purification, formulation, and process development.

Fouling, Cleaning, Cost & Consumables

How does membrane fouling impact protein processing, and what strategies minimize fouling during TFF?
Membrane fouling can reduce permeate flux, increase processing time, and lower protein recovery by causing concentration polarization, aggregation, or deposition of biomolecules on the membrane surface. To minimize fouling during TFF, several strategies can be applied: use low-binding membranes (RC for sensitive proteins), maintain appropriate tangential flow and transmembrane pressure (TMP) to prevent protein compaction, operate at controlled temperature and buffer conditions. These approaches collectively maintain high recovery, reproducibility, and protein stability during processing.

What are the cost and consumable considerations when using TFF for routine protein concentration and formulation?
When using TFF for routine protein concentration and formulation, key cost and consumable considerations include the membrane/filter chips, buffer and reservoir containers, and cleaning reagents. In systems like µPulse and aµtoPulse, the primary consumable is the TFF filter chip, which may be single-use or reused with validated cleaning-in-place (CIP) — and replacement frequency depends on membrane life and sample load. Choosing low hold-up volume chips reduces sample waste and reagent use, lowering overall costs. Automated CIP with NaOH and preservative storage solutions helps extend membrane life and cut consumable turnover. While TFF consumables can be more costly than simple spin filters upfront, the high recovery, reduced waste, faster runs, and reproducibility typically offset costs in routine workflows, especially for precious proteins and scalable formulation work.

Protein Processing

Protein Processing Using the µPulse - TFF System