Mild pH Elution Benefits of Purolite™ Praesto™ Jetted A50 HipH
Overview
Protein A affinity resins are most commonly used to perform the capture step in therapeutic drug purification workflows. However, this step contributes significantly to the overall production cost of protein purification.
Ensuring that maximum yield is obtained during purification is critical. However, antibody-related molecules (primarily bispecifics) experience high levels of aggregation when standard protein A resins are utilized in the elution step due to the low pH. This results in a significantly lower yield of the active material needed.
Purolite Praesto Jetted A50 HipH is designed to allow elution at higher pH levels, minimizing aggregation and reducing the amount of host cell protein (HCP) remaining after the capture step of purification.
In this study, a series of antibody molecules were investigated using Purolite Praesto Jetted A50 HipH and three additional Protein A resins. Resin performance was assessed based on the purity of each antibody and the HCP levels remaining following the first purification step.
Investigation
Two Purolite™ protein A resins, were investigated for purity (content of the desired target molecule monomer) and residual HCP when eluting three different mAb-related molecules. The first resin, Purolite™ Praesto™ Jetted A50, is a native Protein A designed for a wide range of mAb constructs eluting at pH 3.5.
The second resin, Purolite Praesto Jetted A50 HipH, allows for the elution of the bound mAb molecule at a higher pH between 4.5 and 4.8. This higher elution resin is particularly useful for the purification of pH-labile antibodies and related constructs or those that are prone to aggregation at low pH levels.
To form a comparison, two competitor resins were also investigated. Competitor A is a widely used native protein A resin, and Competitor B is an engineered high-performance protein A resin. Both competitor resins elute at pH 3.5.
To evaluate performance across a wide range of antibodies, purity and residual HCP were investigated across three molecules following the first step of purification using each of the four protein A resins previously noted. The three different molecule types used in this study include: Fc-fusion, pH-sensitive mAb, and bispecific.
- Fc-fusion molecule: Antibody where the Fc region is combined with another protein.
- mAb: A standard monoclonal antibody.
- Bispecific: IgG1 bispecific with two binding sites on either antibody ‘arm,’ targeting different molecules.
Across the ‘standard’ Protein A resins, elution was held at pH 3.5 for each molecule type. For Purolite Praesto Jetted A50 HipH, elution was 4.5 for the Fc-fusion, 4.5 for the mAb, and 4.8 for the bispecific.
Figure 1 shows the purity results (amount of monomer), and
Figure 2 shows the residual HCP in the samples.
Conclusion
Across the standard mAb and Fc-fusion, all resins provided a similar yield of the desired biologically-active monomer. However, for the bispecific, prone to aggregation at low pH,
Purolite Praesto Jetted A50 HipH provided a significantly higher yield of biologically-active monomer content. An improvement in yield of approximately 10% leads to favorable process economics in the purification workflow.
This process improvement is also demonstrated by the lower presence of HCPs in the sample when using Purolite Praesto Jetted A50 HipH. A reduction in residual HCPs relieves the strain of further purification steps.
In addition, Purolite Praesto Jetted A50, a ‘standard’ protein A resin, also outperformed competitor resins. This difference in performance can presumably be attributed to the Jetting
manufacturing process which produces more uniform beads compared to the traditional method of emulsification.
In conclusion, the results demonstrate the efficacy of Purolite Praesto Jetted A50 HipH for the purification of pH-labile antibodies prone to aggregation. In this study, Purolite Praesto Jetted A50 HipH proves favorable for process economics — providing greater yield with fewer HCP contaminants.
