Bioconjugate Cleanup

Which MWCO membranes are recommended for bioconjugates?
For bioconjugates like antibody-drug conjugates (ADCs) or protein-polymer conjugates, a membrane with a molecular weight cut-off (MWCO) 2–3 times smaller than the size of the conjugated molecule is recommended. This ensures high retention of the conjugate while allowing smaller impurities (unreacted drugs, linkers, solvents) to pass through. This selection maximizes conjugate recovery, maintains stability during diafiltration, and supports efficient buffer exchange into final formulation buffers. Always validate membrane performance at small scale (e.g., using the µPulse and aµtoPulse systems) to confirm recovery and purity before scaling.

How does TFF ensure removal of small unconjugated molecules while retaining the conjugated product?
TFF ensures removal of small unconjugated molecules while retaining the conjugated product by using a membrane with an appropriate molecular weight cutoff (MWCO) that allows low-molecular-weight species—such as free drug, linkers, or salts—to pass through the permeate, while the larger conjugated biomolecules are retained in the retentate. In systems like µPulse and aµtoPulse, a range of pore sizes, controlled transmembrane pressure, low hold-up volume, and gentle tangential flow maintain high recovery and prevent aggregation, enabling efficient separation of small contaminants without compromising the integrity or concentration of the conjugated product.

How do regenerated cellulose (RC) vs. mPES membranes affect recovery and binding in bioconjugate workflows?
Regenerated cellulose (RC) membranes have low non-specific binding, making them well suited for sensitive bioconjugates where maximal recovery is critical. Modified polyethersulfone (mPES) membranes offer higher flux and chemical resistance, which can be advantageous for viscous samples or demanding buffer conditions, but may have slightly higher non-specific adsorption. Choosing between RC and mPES in systems like µPulse and aµtoPulse depends on the sample type, buffer compatibility, and recovery requirements, ensuring efficient separation of unconjugated molecules while preserving the integrity of the bioconjugate.

Can TFF replace dialysis or SEC in conjugation workflows?
Yes, TFF can replace dialysis or size-exclusion chromatography (SEC) in conjugation workflows. Unlike dialysis, TFF is much faster and allows precise buffer exchange without prolonged incubation, while maintaining high recovery and minimizing aggregation. Compared to SEC, TFF can process larger volumes with better scalability and reduced hands-on time. Automated systems like µPulse and aµtoPulse further enhance this by providing low hold-up volumes, controlled transmembrane pressure, and reproducible recovery, making TFF an efficient, scalable alternative for bioconjugate purification and formulation.

In what scenarios is TFF preferable over normal flow filtration (NFF) or dialysis for bioconjugate diafiltration and buffer exchange?
TFF is preferable over normal flow filtration (NFF) or dialysis when rapid processing is needed, because TFF achieves buffer exchange or diafiltration in minutes while dialysis may take hours. It is also preferred when high recovery of sensitive bioconjugates is critical, since tangential flow minimizes shear, aggregation, and adsorption compared to NFF. TFF is highly scalable, allowing parameters to transfer from small- to pilot-scale without compromising performance. It also provides precise volume and concentration control, and automated systems like µPulse and aµtoPulse maintain stable transmembrane pressure and low hold-up volumes, ensuring reproducible results. Overall, TFF offers faster, gentler, and more controllable purification for bioconjugates compared to traditional NFF or dialysis.

How does TFF compare to chromatography methods (e.g., SEC) in terms of speed, cost, and scalability for bioconjugate cleanup?
TFF offers several advantages over chromatography methods like SEC for bioconjugate cleanup. It is faster, as buffer exchange and concentration can be completed in minutes rather than hours required for column-based separation. TFF is also more cost-effective, reducing the need for expensive resins and minimizing labor-intensive manual steps. In terms of scalability, TFF systems like µPulse and aµtoPulse allow direct transfer of parameters from small-volume lab workflows to pilot- or production-scale processes without compromising recovery or product integrity. While SEC provides high-resolution separation, TFF delivers efficient, reproducible, and gentle purification for a wide range of sensitive bioconjugates with lower operational complexity.

How does automated diafiltration with TFF simplify bioconjugate formulation into the desired buffer?
Automated diafiltration with TFF simplifies bioconjugate formulation by continuously removing unwanted small molecules while simultaneously introducing the target buffer, eliminating multiple manual buffer exchanges. Systems like µPulse and aµtoPulse provide precise transmembrane pressure control, low hold-up volumes, and gentle tangential flow, ensuring high recovery and preserving biomolecule integrity. This automation reduces hands-on time, minimizes sample loss, and delivers reproducible, scalable results for proteins, ADCs, viral vectors, and other sensitive bioconjugates.

Can TFF systems be scaled from research-scale bioconjugate cleanup to GMP-compliant manufacturing?
Yes, TFF systems like µPulse and aµtoPulse enable scalable bioconjugate workflows from research-scale cleanup to GMP-compliant manufacturing. Their controlled transmembrane pressure, low hold-up volumes, and reproducible recovery allow parameters optimized on small volumes to be directly translated to larger-scale systems. Additionally, single-use chips and validated cleaning-in-place (CIP) protocols support regulatory compliance, while maintaining product integrity, homogeneity, and high recovery, making TFF an efficient and scalable solution for both early-stage development and GMP manufacturing of bioconjugates.

What is the minimum and maximum sample volume that can be processed for bioconjugate cleanup using µPulse and aµtoPulse systems?
The µPulse TFF system can efficiently process sample volumes from 1 mL up to 100 mL, while the aµtoPulse system supports a broader range, handling 0.5 mL to 100 mL with a minimum final volume as low as 250 µL. These low hold-up volumes and precise flow controls ensure high recovery and minimal product loss, making both systems well suited for bioconjugate cleanup across small- to medium-volume workflows.

Is single-use TFF preferred for cytotoxic conjugates?
Yes, single-use TFF is preferred for cytotoxic conjugates. It eliminates cross-contamination risks, reduces the need for aggressive cleaning, and ensures operator safety when handling hazardous materials. Systems like µPulse and aµtoPulse support single-use chips, enabling high recovery and reproducibility while maintaining containment and workflow flexibility for cytotoxic or high-risk bioconjugates.

How does site-specific conjugation affect the need for TFF in cleanup, and can it reduce the complexity of removing heterogeneous mixtures?
Site-specific conjugation reduces product heterogeneity, but cleanup is still essential to remove unreacted drug, free linker, and small-molecule byproducts. TFF is ideal in this context because it enables precise buffer exchange and concentration while preserving the integrity of the conjugated biomolecule. By combining low hold-up volume, controlled transmembrane pressure, and gentle tangential flow (as in µPulse and aµtoPulse), TFF efficiently separates small contaminants from the site-specifically conjugated product, simplifying downstream purification and improving overall recovery and reproducibility.

What temperature, pH, or buffer compositions best support TFF performance for shear-sensitive bioconjugates?
To support optimal TFF performance for shear-sensitive bioconjugates, it is recommended to maintain temperatures between 2–8 °C or at ambient conditions suitable for protein stability, and a pH range near physiological or formulation-optimized levels (typically pH 6–8) to preserve conjugate integrity. Buffers should include stabilizers such as low salt, glycerol, or sugars to reduce aggregation, while avoiding high-viscosity or highly charged solutions unless compatible with the selected membrane (RC or mPES). Systems like µPulse and aµtoPulse further enhance performance by providing controlled transmembrane pressure and gentle tangential flow, enabling efficient concentration and diafiltration of sensitive bioconjugates without compromising recovery or stability.

What strategies are available to minimize sample loss or aggregation during TFF of sensitive conjugates?
To minimize sample loss or aggregation during TFF of sensitive conjugates, it is important to combine several strategies. Gentle tangential flow and controlled transmembrane pressure (TMP) reduce shear stress, preserving biomolecule integrity. Selecting low-binding membranes such as RC, or chemically resistant mPES membranes when appropriate, helps minimize adsorption and fouling. Maintaining optimal temperature, pH, and buffer composition, including stabilizers like glycerol or sugars, further prevents aggregation. Using low hold-up volume systems like µPulse and aµtoPulse maximizes recovery, especially for small-volume samples, while automated cleaning-in-place (CIP) and pre-conditioning ensure membranes are free of residual material that could induce aggregation. Together, these measures ensure high recovery, reproducibility, and minimal aggregation for bioconjugates, ADCs, and other sensitive biomolecules.

Can TFF be used for bioconjugates beyond ADCs, such as fluorescently labeled proteins, oligo-protein complexes, or nanoparticle conjugates?
Yes, TFF can be used for a wide range of bioconjugates beyond ADCs, including fluorescently labeled proteins, oligo-protein complexes, and nanoparticle conjugates. Its gentle tangential flow and controlled transmembrane pressure preserve the structural integrity of these sensitive molecules, while automated systems like µPulse and aµtoPulse provide low hold-up volumes, precise recovery, and reproducible buffer exchange. This makes TFF an efficient and scalable solution for purifying, concentrating, and formulating diverse bioconjugate types.