Application note Optimizing bispecific antibody purification: High-resolution polishing with SP PuraBead® Edge Polishing is a critical step in bispecific antibody (bsAb) purification, essential for achieving high purity and yield. It plays a key role in removing process-related impurities—such as host cell proteins (HCPs), DNA, leached Protein A, endotoxins, viruses, and aggregates—as well as productrelated impurities like mispaired antibody chains. Strong cation exchange (CEX) chromatography is commonly used to separate these closely related impurities. This application note demonstrates the use of SP PuraBead® Edge for high-resolution polishing of a bsAb feedstock.
SP PuraBead® Edge is a high-performance strong cation exchange (CEX) chromatography resin designed to deliver highresolution peak separation and flow properties and is therefore well-suited for the bsAb polishing step. The target bsAb binds to SP PuraBead® Edge under low-salt conditions and at a pH below its isoelectric point (pI, typically 6–9 for antibodies), where it carries a positive charge and interacts with the resin’s negatively charged groups. Aggregates, which bind more strongly, can be separated using a salt or pH gradient. This allows the bsAb to elute first, followed by the more strongly retained impurities. The resin features a sulphopropyl (SP) functional group on a 6% cross-linked agarose matrix with a mean particle size of 65 ± 10 µm and delivers a high binding capacity of up to 95 mg/mL (measured with lysozyme at 10% breakthrough). The smaller bead size delivers the high resolution needed for polishing applications where fine separation is critical, such as in bispecific antibody (bsAb) purification. This enables efficient removal of closely related impurities, including aggregates and mispaired chains, while maintaining product integrity. Its chemical stability in up to 1.0 M sodium hydroxide supports stringent cleaning protocols and consistent performance across multiple cycles.
Dynamic binding capacity Dynamic binding capacity (DBC) is a critical factor in evaluating resin performance for efficient antibody purification. SP PuraBead® Edge demonstrated strong and consistent DBC across three different residence times (RT) when tested using a model protein solution of lysozyme at 3 g/L in triplicate (Figure 1). It maintained this consistent binding performance without a significant reduction in capacity, even at higher flow rates. 105.00
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Average DBC (mg/mL of resin)
During bsAb production, mispaired species, such as homodimers, half-antibodies, and mixed light-chain variants can form due to incorrect pairing of heavy and light chains. These closely related impurities are challenging to separate and require high-resolution techniques.
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SP PuraBead® Edge
This application note describes the use of SP PuraBead® Edge for polishing a bsAb feedstock, highlighting its performance in achieving high-resolution separation of mispaired chains.
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Residence time (min) Competitor 34 µm
Competitor 90 µm
Figure 1: Comparison of SP PuraBead® Edge resin dynamic binding capacity (DBC) with two commercially available CEX resins of different particle sizes across different residence times.