Keeping mAb aggregation low during the final UFDF step

mAb aggregation: why and when it happens

Monoclonal antibody (mAb) aggregates are considered an impurity in bioprocessing as they can impact immunogenicity in patients. Aggregates will often form in the cell culture bioreactor, but they can also form during the downstream purification. In downstream processing, shear, thermal, and solution conditions all contribute to stress on the mAb, which can lead to aggregation. By optimizing your equipment, materials, and processes, you can keep aggregates low. Read how to control aggregates during chromatography.

What to consider for final formulation

By the time a mAb reaches this step, the focus usually shifts from removing aggregates to preventing them. At this stage, the mAb is concentrated and exchanged into its final buffer using tangential flow filtration (TFF). During TFF, aggregates can form due to shear exposure at solid-liquid and air-liquid interfaces, thermal stress, or leachables from the system (1, 2, 3). These issues are even harder to control with high-concentration mAbs. Careful TFF system design, material choice, and process development help reduce these risks and keep aggregate formation low.

Factors that influence aggregation

Characteristics of the molecule

Proteins differ in how easily they associate with each other or unfold. Small changes in mAb complimentary-determining regions (CDR) can strongly affect charge or hydrophobic interactions (4, 5, 6). Differences in how flexible the molecule is—in part related to IgG type (6, 7)—can affect unfolding to expose hydrophobic patches, which can lead to aggregation.

Fig 1. Antibody variants - mAb, BsAb, Diabody, dAb

Process conditions

You can reduce the tendency for proteins to self-associate by optimizing their background solution (1, 8). pH and ionic strength are levers that allow control of viscosity and protein–protein interactions (5). Additives, such as sugars and amino acids, can protect proteins from heat and shear stress. And surfactants can help prevent proteins from adsorbing to air or solid interfaces (3, 9).

During ultrafiltration (UF) / diafiltration (DF), the mAb becomes more concentrated, which can increase protein-protein interactions and speed up aggregation. The mAb usually starts in a buffer suited for the previous purification step. Then, it's exchanged into a formulation buffer designed to lower viscosity and improve stability. TFF often follows three steps: concentrate, diafilter, then concentrate again. For diafiltration, it's important to carefully choose the mAb concentration to limit both rising viscosity and aggregate formation.

TFF equipment and materials

Fig 2. Integrated UniFlux 30 filtration system and 100 L tank allow fully automated CFF

Pumps and valves

During UF/DF, the pump can expose the mAb to shear, heat, and interfacial stress. When using transmembrane pressure (TMP) control, the flow-restriction valve can also add shear stressors. Pump friction generates heat that can promote aggregation. In peristaltic pumps, elastomeric tubing deformation and contact between tubing walls can disturb proteins adsorbed to surfaces, leading to aggregate formation (10). Finally, pressure differences across pumps and valves can lead to cavitation. The mAb can adsorb to the hydrophobic surfaces of air bubbles, unfold, and re‑enter the solution to form aggregates. When bubbles collapse, they can also create high local pressure and shear.

TFF cassettes

As proteins flow through the TFF cassette, they face more shear stress. For some mAbs, feed flow rates have been shown to correlate with aggregation rate (11). The screen type also matters. It either directly changes the shear exposure or changes the fluid dynamics, altering the concentration polarization layer at the membrane surface, which alters the level of protein-protein interaction (12).

Recirculation vessels

Recirculation vessel design plays a key role in controlling aggregates during UF/DF. If the return line is too close to or "upstream" of the filter feed line, "short‑circuiting" can create very high local mAb concentrations. And if the return line is at the top of the vessel, splashing can cause foaming and stress at the air-liquid interface. It's also important to monitor mixing rates and fluid levels. When the fluid level drops below the impeller, foaming and interfacial stress can increase. You can reduce these risks by choosing mixers with good turn-down ratios and, at high concentration factors, by using a "feed‑and‑bleed" approach that feeds a larger tank into a smaller recirculation vessel during concentration.

Modern solutions

Most modern UF/DF systems use a combination of valves, tubing size, and pump output to avoid excessive linear velocities through tubing and shear throughout the flow path. Manufacturers also design today's consumables to limit leachables that can drive aggregation. For example, ÄKTA readyflux™ filtration systems work with Cytiva's T-series TFF cassettes and Xcellerex™ XDUO single-use mixers to support gentle, controlled UF/DF processing.

Fig 3. ÄKTA readyflux™ single use TFF systems

Solution for unstable or high-concentration mAbs

For some unstable molecules, tuning process settings and solution conditions isn't enough to stop aggregation. In these cases, you can use hollow‑fiber TFF modules to lower shear in the filter. You can also turn to single‑pass TFF (SPTFF) to reduce shear from the pump, flow-restriction valve, and filter (13).

Fig 4. Cadence™ single pass TFF modules.

SPTFF runs at much lower crossflow rates and concentrates or diafilters the product in one pass through the filter. It removes the need for a recirculation vessel, which shortens mixing time of the concentrated molecule. You can also use SPTFF after a standard UF/DF step to reach final drug concentration while limiting shear for high-concentration formulations or avoiding very low volumes in the recirculation vessel. Read about optimizing TFF and SPTFF for high-concentration mAbs.