Annika Söderholm, Scientist, Cytiva
Martin Sjöberg, Global Product Manager, Cytiva
Monoclonal antibodies (mAbs) and antibody variants are a cornerstone of modern biotherapeutics, and producing them to levels of purity and quality required for therapeutic products is essential for manufacturers. Doing so involves careful optimization during both upstream and downstream process development.
Even when cell culture conditions are optimized, impurities that may need to be removed in downstream purification include antibody aggregates, mispairs, charged variants, viruses, host cell proteins (HCP), and endotoxins—in addition to cell culture medium components.
Among the product-related impurities encountered, aggregates are particularly problematic. Aggregates can form at many points in a process—including cell culture, capture, polishing, and even filtration—because antibodies may be sensitive to shifts in pH, concentration, shear, and other process conditions. Antibody fragments and bispecific antibodies are often more prone to form aggregates (Fig 1). These aggregated species matter because they reduce product efficacy and can increase the risk of immunogenicity. While levels in harvest pools can range widely, from about 1% to 15%, the aim in downstream processing is generally to reach less than 1%, to reduce the risk of adverse effects and regulatory concerns.
Addressing aggregation isn’t straightforward: aggregates often share many physicochemical properties with the monomer, making separation difficult during downstream purification. Process developers therefore rely on a combination of strategies to prevent aggregation and to remove it efficiently when it occurs. In this article we’ll share downstream chromatographic strategies that we’ve used successfully to remove aggregates. Our recommendations are based on studies conducted in our labs, and we’ve included links to the full studies so you can read them in detail.
Filtration steps can also affect aggregation. Check out this article on tangential flow filtration (TFF) strategies that can help reduce aggregation risk during concentration and buffer exchange.
Fig 1. Aggregate created in the feed due to mispairing of a bispecific antibody containing a single-chain variable fragment (scFv). ScFv has a strong tendency to aggregate.
What are mAb aggregates and why are they hard to remove?
mAb aggregates are a type of product-related impurity. They can be dimers, trimers, or multimers of the antibody molecule. Dimers are typically the most common type of antibody aggregation, and the most challenging to remove. We’ve conducted various studies on aggregate removal, which we summarize here. You’ll find key details on what we’ve learned as well as links to the full study details.
Typical mAb chromatography processes
mAb purification typically begins with an affinity capture step using a protein A or protein L resin. It is followed by either one or two polishing steps—e.g., a single multimodal anion exchange step in flow-through mode, or a cation exchange polishing step in bind-elute mode followed by an anion exchange step in flow-through mode (Fig 2).
Fig 2. The Cytiva mAb toolbox for 2- or 3-step purification. These protocols are a good starting point for purifying mAbs.
These baseline protocols work well for many mAbs, but not all of them. For “tricky” antibodies that aren’t sufficiently purified with these strategies, you’ll need to adapt—e.g., by changing buffers or using different polishing techniques or resins.
Antibody aggregation in the capture step
Aggregation often occurs upstream during cell culture, but it can also happen, for example, during capture when the mAb concentration is high and the elution buffer pH is low.
There are ways to prevent aggregation during capture. Here are some examples:
- For pH-sensitive targets, you can try a capture resin designed for elution at higher pH than is typical for mAbs. An example is MabSelect™ mild elution resin.
- Adding an excipient such as arginine to your buffer can help prevent on-column aggregation.
- If you’re using a protein L resin such as MabSelect™ VL resin, changing the elution buffer from citrate to propionate or succinate can substantially increase the elution pH, which can help prevent aggregation. Read this article to learn more.
Furthermore, aggregates generally bind strongly to the protein A resin and tend to elute in the tail, sometimes even creating a visible shoulder on the elution peak. To remove aggregates in the capture step, you can try running a short (5 column volume (CV)) pH gradient and/or using more stringent peak collection.
Polishing strategies to solve aggregate removal challenges
Aggregates generally show stronger binding to ion exchange (IEX) and hydrophobic interaction chromatography (HIC) resins than the monomeric target molecule. You can exploit this property during polishing steps to achieve effective separation. Multimodal resins, which combine both IEX and HIC functionalities, are particularly efficient in such cases, because they offer enhanced selectivity for aggregates (Fig 3). Capto™ adhere resin, for example, was specifically engineered to enable aggregate removal for mAbs in flow-through mode (see example 2 below).
Fig 3. Our recommendations for tweaking the polishing strategy if you face challenges with aggregate removal.
Below is a list of polishing resins that we suggest for processes where mAb aggregate removal is challenging.
| Polishing step 1 | Polishing step 2 |
|---|---|
| CIEX resins:
- Capto™ S ImpAct resin - Capto™ SP ImpRes resin HIC resin: - Capto™ Phenyl (high sub) resin |
Multimodal resins:
- Capto™ adhere ImpRes resin (for bind/elute mode) - Capto™ adhere resin (for flow-through mode) |
Note: End users must always ensure freedom to operate for the specific processes used for purifying their specific molecules.
Using HTPD and statistical or mechanistic modeling methods to optimize faster
To optimize aggregate removal while achieving high purity and yield, high-throughput techniques are widely used. These parallel and miniaturized approaches allow rapid screening of multiple conditions with minimal material, generating rich data sets. Often combined with statistical models such as design of experiments (DoE), these methods help identify key factors for aggregate clearance. For even deeper understanding and extended predictive capability, mechanistic modeling offers a powerful way to simulate process behavior and identify optimal conditions for aggregate removal.
Application examples
Example 1: Using Capto™ S ImpAct CIEX resin for the first polishing step
In the first example, a mAb sample contained 7% aggregation after protein A capture. We processed the sample using Capto™ S ImpAct CIEX resin in gradient elution mode. The goal was to reduce aggregate content to < 1% while maintaining monomer recovery above 90%. Buffer screening identified strong binding at pH between 5 and 6 with slightly higher ionic strength (Table 1). However, resolution of the monomer from aggregates was better at pH 5.0. A binding buffer of 50 mM sodium acetate and 50 mM NaCl pH 5.0 and a 20 CV elution gradient to 500 mM NaCl achieved good results both in terms of dynamic binding capacity (DBC) and monomer/aggregate selectivity (Fig 4). Under these buffer conditions and at a mAb load of 50 mg/mL resin, aggregates were reduced from 7% to 0.7%, representing a tenfold reduction, while achieving more than 90% yield. To maximize product yield, we used a DoE approach to optimize elution conditions. Adjustment of flow rate during elution gave us a robust process, maintaining high purity and yield even at a mAb load of 80 mg/mL.
Read the full study: Polishing of monoclonal antibodies using Capto™ S ImpAct resin.
Table 1. DBC of Capto™ S ImpAct resin at different ionic strengths and pH values
| Buffer | DBC (mg/mL) |
|---|---|
| 50 mM sodium acetate, pH 5.0 (low IS) | 71 |
| 50 mM sodium acetate, 50 mM NaCl, pH 5.0 | 109 |
| 50 mM sodium acetate, 50 mM NaCl, pH 5.5 | 122 |
| 100 mM sodium acetate*, pH 6.0 | 124 |
| 50 mM sodium acetate, pH 6.0 (low IS) | 89 |
*Buffer capacity was too weak to add 50 mL NaCl at pH. Ionic strength was instead increased by increasing acetate concentration.
Fig 4. Cumulated aggregates vs cumulated mAb monomer recovery after gradient elution at different pH values. IS, ionic strength of buffer.
Example 2: Using Capto™ adhere multimodal AIEX resin for mAb polishing
The second example involves a mAb sample containing approximately 6% aggregate after protein A capture. In this case, we used Capto™ adhere resin, a multimodal anion exchanger, in flow-through mode. Screening determined that a pH of 6.5 and conductivity of about 30 mS/cm (equivalent to 300 mM NaCl) provided optimal conditions. Under these conditions and at a sample load of 120 mg/mL, aggregate content was reduced from 6% to less than 1%, while maintaining yields above 90%. Even at the highest load tested, 265 mg/mL, this method delivered good reduction of dimers and aggregates (Figs 5 and 6).
Read the full study: Selective removal of aggregates with Capto™ adhere resin.
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Experimental conditions
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Fig 5. Polishing on Capto™ adhere resin
Fig 6. Dimer and aggregate (D/A) content in starting material (0 = load) and fractions collected during sample loading.
Additional examples
- Using Capto™ SP ImpRes resin for the first polishing step
Aggregate content was reduced from ~11% to <1% by optimizing conditions with Capto™ SP ImpRes resin. - Using Capto™ adhere ImpRes resin in bind and elute mode
Aggregate content reduced from ~2.4% to 0.5% by optimizing conditions with Capto™ adhere ImpRes resin. - Using Capto™ Phenyl (high sub) for aggregate removal
Aggregate reduced from ~3.5% to <1% by flow through step with Capto Phenyl (high sub) resin. - Using Capto™ Butyl ImpRes resin for aggregate removal
Aggregate reduced from ~10% to <1% by grained or isocratic elution with Capto™ Butyl ImpRes resin.
Preventing aggregation with TFF
During the UF/DF step, the mAb is concentrated and exchanged into its final formulation buffer using TFF. During the process, protein aggregation can be induced by shear exposure at solid-liquid and/or air-liquid interfaces, thermal stress, or leachables from the system. Learn more about aggregation prevention using UF/DF here.
Conclusion
Removing aggregates in mAb processes can be challenging due to their similarity to the target molecule. This guide presents chromatographic strategies for aggregate control. Optimizing the capture step can support both the prevention and removal of aggregates, while polishing methods using CIEX, HIC, and multimodal resins—supported by high‑throughput screening and modeling tools such as DoE or mechanistic modeling—offer powerful separation options even for challenging molecules.
Additional resources:
- Polishing chromatography in process development: a complete guide
- Quick guide: setting up downstream processes for mAbs and antibody variants
- Explore chromatography resins
- Developing a HIC polishing step for removal of mAb aggregates
- How to optimize a HIC step with HTPD and DoE
- Optimization of an antibody polishing step with mechanistic modeling
- Webinar on demand: Optimizing your mAb capture and polishing with the latest resins and tips
- DoE resource center
- Mechanistic modeling resource center