Purification Techniques | How to Creatively Remove Aggregates?

Dr. William Hallett from the FDA delivered a keynote presentation at the 2016 CHI Annual Conference, discussing the impact of protein aggregates on immunogenicity. The presentation highlighted that protein aggregates can form during the production process, product transportation, storage, and delivery. Due to the potential immunogenicity of aggregates, controlling their levels is considered a critical quality attribute.

With the rapid development of antibody drugs, the targeting of antibodies has shifted from single targets to multiple targets. The trend in antibody development has also become more diverse, with a focus on innovative drugs using technologies such as bispecific antibodies and antibody-drug conjugates (ADCs). This diversified approach to drug development presents unprecedented challenges for downstream purification processes. The removal of impurities such as aggregates has become a crucial process hurdle. In this article, we will share strategies for removing aggregates using different chromatographic principles.

Protein A affinity chromatography MaXtar ARPA: Additive-assisted aggregate removal.

Protein A affinity chromatography products are widely used in the capture step of antibody purification. As protein A specifically binds to the Fc region of antibodies, this step can significantly reduce process-related impurities, including host cell proteins and nucleic acids. However, under typical conditions, protein A chromatography has poor efficiency in removing aggregates. It is known that aggregates have stronger binding affinity than monomers, and adjusting the elution pH alone is often insufficient for achieving good separation. Therefore, in many cases, the removal of aggregates relies on subsequent ion exchange chromatography or multimodal chromatography. In these cases, the addition of calcium chloride/polyethylene glycol (PEG) or sodium chloride/PEG combinations to the wash and elution buffers can significantly enhance the ability of protein A to remove aggregates. The synergistic effect of salt and PEG promotes effective separation of monomers and aggregates.

Sample Information: clarified culture fluid

Equilibration Buffer: 50 mM Tris-HAc, 150 mM NaCl, pH 7.4

Washing Buffer: 50 mM NaAc-HAc, 500 mM CaCl2, 5% PEG, pH 5.5; or 50 mM NaAc-HAc, 750 mM NaCl, 5% PEG, pH 5.5

Elution Buffer: 50 mM HAc, 500 mM CaCl2, 5% PEG, pH 3.1; or 50 mM HAc, 750 mM NaCl, 5% PEG, pH 3.1

Figure 1: Protein A purification chromatogram with the addition of 500 mM CaCl2 and 5% PEG.

Figure 2: Overlay chromatograms of Protein A chromatography under non-optimized and optimized Conditions.

Summary: As observed in Figure 1 and Figure 22, the addition of 500 mM CaCl2 and 5% PEG, or 750 mM sodium chloride and 5% PEG to the wash and elution buffers, significantly improves the separation of monomers and aggregates, leading to a noticeable improvement in SEC purity.

Multi-modal anion exchange chromatography MaXtar MMA: Removing aggregates through flow-through mode.

MaXtar MMA is a strong anion-exchange multimodal resin, where the ligands interact with the target molecules through various mechanisms such as ion exchange, hydrophobic interactions, and hydrogen bonding. MMA is commonly used in the purification stage of antibody purification, allowing for the removal of aggregates, protein A ligands, host cell proteins (HCPs), DNA, and viruses, among other impurities.

Sample Information: Dual antibody Protein A elution buffer, with 11% aggregate content Equilibration Buffer: 50 mM Tris-HAc, pH 7.5 Sample

Sample Loading: Protein A elution buffer (pH adjusted to 7.5) (loading capacity 80 mg/ml)

Washing Buffer: 50 mM Tris-HCl, pH 7.5

Figure 3: MaXtar MMA Chromatogram

Summary: SEC results indicate that the aggregate content of the dual antibody decreased from 11% to 1.6%.

Product Link: https://www.biolink.com/products/maxtar-mma-multimodal-chromatography-resins/

Multi-modal cation exchange Chromatography MaXtar MMC HR: Removing aggregates through binding mode.

MaXtar MMC HR is a multi-mode weak cation exchange resin used for the fine purification of antibodies. It is designed to effectively remove impurities such as aggregates and degradation fragments.

Product Link:https://www.biolink.com/products/maxtar-mmc-multimodal-chromatography-resins/

Figure 4: Multi-mode chromatographic atlas

Figure 5: SEC (Size Exclusion Chromatography) detection chromatogram

Hydrophobic chromatography: Removal of aggregates.

Aggregates have stronger hydrophobicity compared to monomers. Choosing a high-resolution MaXtar Phenyl HR can efficiently remove aggregates.

Figure 6: High-resolution elution chromatograms of Phenyl under non-optimized and optimized conditions.

Summary:  Figure 6 shows that the elution purity of Phenyl can reach above 98%. Additionally, the yield can be improved by adding hexylene glycol/arginine. For bispecific molecules with strong hydrophobicity, it is recommended to choose less hydrophobic stationary phases such as MaXtar Octyl/Butyl to allow the bispecifics to flow through while retaining the aggregates.

Product Link: https://www.biolink.com/products/maxtar-hr-hydrophobic-chromatography-resins/