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.
Typically, we use a pH of 3.5 to 4.0 to elute antibodies in the protein A chromatography capture step. However, some antibodies and antibody-derived molecules aggregate at this low pH—specifically, molecules like bsAbs (containing an Fc region), IgG4, and Fc fusion proteins. Aggregation can compromise monomeric purity and yield, placing additional pressure on the following polishing steps.
MabSelect™ mild elution resin is one of Cytiva’s protein A resins designed to address various purification needs and minimize the risk of molecules being rejected because of inadequate purification.
In this article, we’ll present studies showing how you can use MabSelect™ mild elution resin in the capture of monoclonal antibodies with elution pH around 5.0.
MabSelect™ mild elution resin: part of the mAb resins toolbox from Cytiva
MabSelect™ mild elution resin is a part of our monoclonal antibody (mAb) resin toolbox, providing a range of capture and polishing resins for clinical and commercial-scale mAb production to fit the diversified antibody pipeline.
To support use in the lab to commercial-scale production, we offer our MabSelect™ mild elution resin in formats for a wide range of scales, good alkaline stability and high DBC, and we supply regulatory support files.
High DBC enables greater antibody processing per unit of resin volume, as well as benefitting productivity and process economy.
For mAb, at a 6 min residence time (RT) and QB10%:
- MabSelect™ mild elution resin showed a DBC of 60 g/L.
- MabSelect PrismA™ resin showed a DBC of 72 g/L.
However, for pH-sensitive molecules, the benefits of higher elution pH may be more important. MabSelect™ mild elution resin can be cleaned using 0.25 M to 0.5 M NaOH for 100 cycles for robust resin CIP.
We based our MabSelect™ mild elution resin on a high-flow agarose base matrix using a median particle (bead) size of 60 μm with excellent flow properties. The resin’s working flow velocity is up to 300 cm/h at 20 cm bed height in large-scale columns.
MabSelect™ mild elution resin interacts specifically with the Fc region, which also allows it to help remove product-related impurities from asymmetric bispecific antibodies (bsAbs) that have one Fc region where protein A binding has been eliminated.
Purification performance in mild elution conditions
Aggregation during monoclonal antibody purification can lower yield and process efficiency. Maintaining a higher elution pH helps reduce acid-induced aggregation and improving product quality.
We investigated the purification performance of MabSelect™ mild elution resin by studying yield, pool volume, mAb aggregates, clearance of host cell protein (HCP), and leached ligand.
We first determined the elution pH of the antibody at a low load to obtain sharp peaks. We used pH gradient elution, which helps identify ideal elution conditions during process development. Once we identified the elution pH, we used the measurement for a step elution to increase resolution and fulfill manufacturing requirements.
We loaded 5 mg trastuzumab (low load) using Tricorn™ 5/50 (1 mL) columns packed with either MabSelect™ mild elution resin or MabSelect PrismA™ resin. Elution took place using a 20 column volume (CV) pH gradient, starting from a buffer of 25 mM sodium acetate and 25 mM sodium phosphate at pH 7.0, and ending with a final pH of 3.4.
Typically, we use sodium citrate in a pH gradient run; but its buffering capacity at the desired pH (pH 7) is limited. As a result, we opted for a phosphate-acetate mixed buffer. Alternatively, a sodium citrate buffer with a pH range of 6.6 to 3.4 can serve as a more straightforward solution for elution gradients.
Figure 1 presents data from this experiment, with our results demonstrating that:
- mAb elutes from MabSelect™ mild elution resin at a pH greater than 5
- Compared to the elution pH of 3.9 observed with MabSelect PrismA™ resin.
Fig 1. Gradient elution (pH 7.0 to 3.4) of a low load of trastuzumab on Tricorn™ 5/50 columns packed with MabSelect™ mild elution resin or MabSelect PrismA™ resin. The mAb eluted at pH > 5 when using MabSelect™ mild elution resin, and at pH 3.9 when using MabSelect PrismA™ resin.
After the pH gradient run, we performed a step elution with the resin loaded to 80% of QB10%. In this experiment, we loaded 36 mL of clarified cell culture supernatant containing 2.46 g/L trastuzumab at 6 min RT on a Tricorn™ 5/100 (2 mL) column. The intermediate wash was performed with 20 mM sodium phosphate, 500 mM NaCl, pH 7.2.
To remove salt, we washed the column with 20 mM sodium phosphate, pH 7, and the bound mAb was eluted using 50 mM sodium acetate, pH 5. This purification process yielded a 97% pure mAb (Fig 2, Table 1).
Fig 2. Chromatogram of purification of trastuzumab using a Tricorn™ 5/100 column packed with MabSelect™ mild elution resin and a step elution at pH 5.0. The target antibody elutes in the main peak with baseline separation and impurities elute in the strip.
The elution pool volume was < 2 CV, and in line with other MabSelect™ resins. It contained < 1% aggregates, 8.3 ppm of leached ligand, and a 3-log reduction of HCP down to 95 ppm (Table 1).
We measured ligand leakage and HCP using Gyrolab technology (Gyros Protein Technologies Group). For ligand leakage, we used a commercial chicken anti-protein A polyclonal IgY antibody (Abcam), and for HCP analysis, we used CHO-HCP kits.
Table 1. HCP, leached ligand, and mAb aggregates in the elution pool in purification of mAb from cell culture supernatant using MabSelect™ mild elution resin
| Load | 80% of DBC at QB10% |
| Yield (%) | 97 |
| Pool volume (CV) | 1.8 |
| Pool concentration (mg/mL) | 23.4 |
| Aggregates (%) | 0.8 |
| HCP* (ppm) | 95 |
| Protein A (ppm) | 8.3 |
*HCP at start: 88 734 ppm
HCP reduction
Conventional protein A affinity chromatography's low-pH elution can lead to coelution of HCP with the target molecule. We explored whether MabSelect™ mild elution resin would provide a lower concentration of HCP in the elution pool compared to a more conventional protein A resin.
We measured HCP levels in elution pools following purification with either MabSelect™ mild elution or MabSelect PrismA™ resin. Resins were packed into Tricorn™ 5/100 columns and clarified cell culture supernatant was loaded onto the columns to 80% of QB10%. Chromatography and HCP analyses followed previous methods, with the MabSelect PrismA™ resin eluted at pH 3.5.
Yields were similar for both resins, but the sample purified with MabSelect™ mild elution resin had a much lower HCP concentration than the MabSelect PrismA™ resin (Fig 3), showing that MabSelect™ mild elution resin removes HCP more effectively.
Fig 3. Remaining HCP (ppm) in elution pool. Concentration of HCP in the loaded feed was 117 443 ppm. Data presented as mean ± SD from triplicate runs. Statistical significance between the two resins was evaluated using a Tukey HSD (P = 0.015).
High elution pH across different mAb classes
We purified three different human IgG subclass molecules—trastuzumab (IgG1), erenumab (IgG2), and nivolumab (IgG4)—to investigate their elution pH on MabSelect™ mild elution resin. As Figure 4 shows, the elution pH was high for all three mAbs, ranging from 5.25 (for IgG2) to 4.8 (for IgG4).
Fig 4. Chromatograms show a pH gradient elution (pH 7.0 to pH 3.4) for three different mAbs, trastuzumab (IgG1), erenumab (IgG2), and nivolumab (IgG4) using MabSelect™ mild elution resin.
We also carried out a step elution for these three mAbs (data not shown) and examined how different elution pH levels affected both pool volume and yield (see Fig 5). Keep in mind that if the elution pH is too high for your molecule, you might end up with larger pool volumes and lower yields.
Optimizing elution pH is key for downstream steps, while balancing pool volume with yield is important. Increased elution volumes lead to less concentrated samples and larger process volumes, which can negatively impact the production facility.
Fig 5. Pool volumes measured in CV (left axis) and yields (right axis) for three human IgG subclass molecules. We used a step elution at different pH values.
Conclusions
We evaluated our MabSelect™ mild elution resin for its ability to purify monoclonal antibodies under conditions that minimize aggregation, improve yield, and process economy. By using gradient elution to identify optimal pH and step elution for the final process, we were able to purify trastuzumab at pH 5.0 with high resolution to 97% yield and 99% monomeric purity.
The process achieved < 2 column volumes in pool size, < 1% aggregates, 8.3 ppm leached ligand, and a 3‑log reduction of host cell proteins (HCP) down to 95 ppm.
We offer our MabSelect™ mild elution resin as bulk resin in units of 25 mL, 200 mL, 1 L, 5 L, and 10 L, and in prepacked column formats such as HiTrap™ columns (1 and 5 mL), HiScreen columns, and on request in production-scale ReadyToProcess™ columns. These formats enable use from lab scale to commercial scale manufacturing and adds to the mAb resin toolbox from Cytiva.
FAQs
What problem does MabSelect™ mild elution resin solve?
Some antibodies and Fc-containing formats aggregate when exposed to the low pH (about pH 3.5 to 4.0) typically used during elution in the protein A step. MabSelect™ mild elution resin lets you elute at a higher pH (around pH 5.0) to help reduce acid-induced aggregation, protect monomeric purity, and improve downstream efficiency.
Which molecules benefit most from mild elution?
Formats such as IgG4, Fc fusion proteins, and asymmetric bispecific antibodies (bsAbs) often show sensitivity to low pH. If your molecule aggregates, loses yield, or requires extra polishing after capture, this resin can be suited for your process.
How does the resin perform compared to MabSelect PrismA™ resin?
While MabSelect PrismA™ resin offers higher DBC, MabSelect™ mild elution resin provides significantly higher elution pH. For trastuzumab, the MabSelect™ mild elution resin eluted the mAb at pH > 5.0 compared to pH 3.9 with MabSelect PrismA™ resin, helping reduce aggregation and improve product stability.
Does the higher elution pH affect impurity removal?
No—you still achieve strong impurity clearance. In fact, studies showed lower HCP levels in the elution pool compared with a traditional protein A resin. The resin also demonstrated 3-log reduction of HCP and low leached ligand levels.
Can I use MabSelect™ mild elution resin in large scale manufacturing?
Yes. The resin is available in multiple bulk formats (25 mL to 10 L) and in prepacked formats such as HiTrap™ columns, HiScreen™ columns, and ReadyToProcess™ columns on request. Its alkaline stability (cleaning with 0.25 M to 0.5 M NaOH for up to 100 cycles) supports commercial-scale robustness.
What dynamic binding capacity (DBC) can I expect?
At a 6 min residence time, the resin shows a DBC of 60 g/L. This supports efficient processing and good productivity while enabling mild elution conditions.
Does a higher elution pH affect pool volume?
It can. If the elution pH is set too high for your specific molecule, pool volume may increase and yield may drop. That’s why pH optimization—usually starting with gradient elution—is important before locking in your step-elution conditions.
What bed heights and flow rates can I use?
The resin is based on a high-flow agarose matrix with a median particle size of 60 µm. You can run up to 300 cm/h at 20 cm bed height in large-scale columns.