Introduction
Process intensification is increasingly vital in biomanufacturing, and includes increasing titers, faster runs, and more cycles to boost productivity. Understanding where intensification enhances value and where it risks process robustness is crucial. In this article, we explore how intensifying chromatography in protein A protocols presents unique challenges (such as increased fouling) when monoclonal antibody (mAb) residues aren’t adequately removed, leading to process deviations.
Understanding fouling in protein A chromatography
Several factors contribute to fouling, including:
- stability of the target antibody
- upstream conditions
- quality of cell culture supernatant post-depth filtration
- resin capacity
- steps of the chromatography protocol
Early stage fouling can be difficult to see, but as it progresses, we can detect it through increased column pressure and elution peak broadening. Resin discoloration can be a sign of fouling─but it can also appear because of other factors.
Fouling is often caused by residual mAb not fully eluted from the column, as well as mAb aggregates, host cell proteins, and product-related variants formed during processing (Fig 1). If these species remain on the resin after elution, they can denature during sodium hydroxide (NaOH) exposure and become difficult to remove.
If denatured protein remains on the column, it can lead to a build-up of protein. While not always visible immediately, the effects often appear after multiple cycles. It’s critical to thoroughly remove mAb from your column before NaOH exposure during cleaning in place (CIP)─not only to avoid fouling—but also to reduce risk of microbial contamination.
Fig 1. Fouled resin (A), resin cleaned with 0.1 M NaOH for 40 min (B), and resin cleaned with 0.1 M NaOH for 3 h (C) analyzed with an electron microscope. Fouled resin has not been exposed to the CIP step.
How process intensification causes fouling
Changing your process conditions to intensify the process can expose protein A resins to new fouling risks. Over time, these changes can compromise resin performance. For example, modifying the composition of your cell culture supernatant loaded onto the capture step can negatively impact the protein A step. Additionally, optimizing your upstream process for higher titers involves increased cell densities, which requires higher capacity in the protein A step and may require more antifoam or switching to a more effective antifoam. While intensifying the depth filtration step can boost your throughput, an increased turbidity may affect the capture step.
Therefore, it’s essential to evaluate the process holistically to identify and mitigate fouling risks. Taking a holistic approach to process intensification can help safeguard the resin, strengthen process robustness, and benefit the mAb process (Fig 2).
Fig 2. Key steps in mAb production. Taking a holistic approach to process intensification helps protect resin performance and support process robustness.
Chromatography protocols are designed to balance purity, yield, efficiency, and economy, with each step serving a specific purpose. Any change to these steps requires careful evaluation, as fouling can develop gradually over repeated cycles and make it difficult to identify when it starts to limit performance.
When fouling begins to impact performance, the instinctive response is often to intensify your cleaning. This might involve increasing NaOH concentration, contact time, or cleaning frequency. However, these methods can introduce new risks and potentially shorten the lifespan of your resins if applied without understanding the underlying fouling mechanisms.
Early warning signs are subtle
Early signs of fouling typically include increased pressure and peak broadening. Capacity loss can also occur, but this isn’t always noticeable if the column isn’t fully loaded. HETP (Height equivalent to a theoretical plate) and asymmetry tests performed over time can help detect fouling, as an increase in HETP or a change in asymmetry indicate declining column performance.
More severe fouling can be seen by low flow zones, lumps, and resin discoloration.
How to avoid fouling
To prevent fouling and maintain optimal performance for your process, we recommend implementing cleaning strategies based on a thorough process and resin understanding. Not all fouling behaves the same, and a one-size-fits-all cleaning approach won’t always deliver sustainable results, especially with the current diverse molecular pipeline and strive to intensify the process.
Understanding which foulants dominate in each process and how they interact with your resin is important. Common contributors include:
- target antibody
- host cell proteins
- aggregates
- product related variants that bind with different strengths
- particulates
You’ll need to ensure all mAbs are removed from the column before cleaning, monitor upstream and downstream process variables, and regularly inspect columns for early signs of fouling.
To efficiently remove mAb and residues of mAb prior to the CIP, we recommend stringent use of an acidic strip (Fig 3) with 0.1 to 0.5 M acetic acid post the elution step. After the acidic strip, a harsh CIP step can be used by balancing NaOH concentration, contact time, flow rate, and direction of flow, while considering what resins can withstand according to vendor specifications.
Fig 3. Protein A purification cycle with its different steps. Include an acidic strip to remove residual mAb before NaOH exposure to avoid fouling.
Harsh cleaning conditions whilst mAb residue remains on the column increases the risk of fouling (Fig 4). We most commonly see fouling at the top of the column (Fig 5), so it’s worth considering running CIP and potentially the acidic strip, in the opposite flow direction to the other process steps. During cleaning, we recommend applying a constant flow over static hold time, as this can improve contact between the cleaning solution and resin bed, supporting more effective removal of residual material.
Fig 4. Resin cleaning effect of acidic strip and different NaOH concentrations at 7.5 min contact time.
Fig 5. Effect of different CIP conditions and protocols. The top and bottom of the columns were analyzed for remaining mAb after the last cleaning cycle. Resin at the top of the column is more fouled than at the bottom.
Cleaning isn’t the only factor affecting performance and fouling risk; the full chromatography process can also contribute. For example, adding salt during elution can reduce its efficiency by increasing conductivity and thereby the risk of fouling. Your choice of buffer matters, and you should carefully evaluate wash steps before shortening or removing them.
Taking an efficient approach to your cleaning, leads to more predictable recovery of dynamic binding capacity and pressure‑flow performance over the entire resin lifetime, supporting intensified chromatography steps, cost control, and environmental impact.
Learn more about cleaning efficiency in our web article A high-throughput study of sodium hydroxide cleaning efficiency for protein A resin and MabSelect PrismA™ resin: Cleaning in place with 0.5 M NaOH.
Restoring performance without unnecessary stress
If fouling occurs, resin performance can often be restored by unpacking the column, treating the resin with NaOH, and repacking it once cleanliness has been confirmed. Visual inspection and electrophoresis analysis of the beads can help verify cleaning effectiveness, while SDS-PAGE can be used to assess residual material on the resin.
Summary
Process intensification can improve your productivity in protein A chromatography, but it must be supported by a cleaning strategy that matches the new process conditions, and should be studied in process development. Fouling often develops gradually and might first appear as increased pressure or peak broadening, and then as loss in capacity or resin discoloration.
A robust approach is to evaluate your full process and, importantly, ensure thorough removal of residual mAb before NaOH exposure. With the right balance of process understanding, monitoring, and cleaning, you can reduce fouling risk, recover performance, and support more predictable resin performance over time.
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FAQs
Why does risk of protein A fouling increase during process intensification?
Higher titers and attempts to reduce process time may introduce more impurities and less efficient cleaning, accelerating accumulation of foulants if cleaning strategies are not adapted.
Can efficient cleaning extend protein A resin lifetime?
Yes. Remove mAb residues in an acidic strip followed by robust CIP helps prevent fouling and maintain resin capacity over the resin lifetime.
When should cleaning strategies be re‑evaluated?
Any major change in upstream conditions, filtration steps, productivity targets, or observed performance trends should trigger a reassessment of cleaning approaches. As the antibody pipeline has diversified, new challenges in purification and stability have emerged. A one-size-fits all approach no longer suffices for purification of antibodies, so there’s a need for a resin toolbox to target individual purification challenges.