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.
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.
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.
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.
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.
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.
SP PuraBead® Edge was compared against two commercially available CEX resins with differing bead sizes. While the 34 µm resin maintained its binding capacity, it could not be tested at the shortest 1.5-minute residence time due to excessive backpressure. The 90 µm resin, on the other hand, showed a drop in binding performance at shorter RTs, highlighting its limitations under high-flow conditions. In contrast, SP PuraBead® Edge delivered consistent performance across all tested RTs, demonstrating superior flow properties and binding efficiency—making it a robust choice for high-throughput purification workflows.
Resolution
Resolution is crucial in chromatographic separations because it determines how well feedstock components, such as mispaired bsAbs, are distinguished from each other in a mixture. Higher resolution ensures more accurate identification and quantification of individual components, reducing the risk of overlapping peaks.
Figure 2 compares the ability of SP PuraBead® Edge and other commercially available CEX resins to resolve a standard protein mix—lysozyme, α-chymotrypsinogen A, and cytochrome C—each loaded to 10% of the resin’s binding capacity. SP PuraBead® Edge demonstrated strong resolving power, successfully resolving peak 2 and peak 3 at all residence times tested. At a 4-minute RT, it also resolved peak 1 and peak 2, though it fell just short of the >1.5 Rs threshold required for full resolution at faster flow rates.
The 34 µm resin achieved full resolution of all three peaks but was limited to residence times above 2 minutes due to excessive pressure. In contrast, the 90 µm resin failed to resolve any peaks when tested at higher flow rates (≤2-minute RT). These results confirm SP PuraBead® Edge’s superior peak resolution and consistent performance across a range of flow conditions.
Peak 1 vs. Peak 2 resolution
PuraBead® Edge Competitor 34 µm Competitor 90 µm Target Residence time (min) SP PuraBead® Edge Competitor 34 µm
time (min)
Peak 2 vs. Peak 3 resolution (B) SP PuraBead® Edge 4-minute RT
Figure 2: Resolution of three proteins achieved using SP PuraBead® Edge resin compared with two commercially available CEX resins of different particle sizes at different residence times (A). Produced chromatogram of SP PuraBead® Edge resolution at a 4-minute residence time (B).
Parameter Description
Adsorbent: SP PuraBead® Edge
Column size: 20 cm bed height in 5 mm diameter housing (3.9 mL CV)
Equilibration buffer: 50 mM sodium acetate, pH 5.0
Elution buffer: 1 M NaCl, 50 mM sodium acetate, pH 5.6
Load volume: 37 mL (46 mg) of Protein A-purified bispecific feed
Elution gradient: 40 CV elution linear gradient from 0–50% elution buffer
Table 1: Initial linear gradient elution conditions for a highaggregate bsAb feedstock using SP PuraBead® Edge.
Resolution of mispaired bispecific antibody chains
Plamotamab is an oncolytic CD20 x CD3 targeting bispecific antibody of growing interest due to its ability to redirect T cells toward tumor cells for targeted killing. Plamotamab has a molecular weight of 125 kDa, consisting of a 25 kDa kappa light chain, a 50 kDa IgG heavy chain, and a 50 kDa chimeric SCFV-heavy-lambda chain. Unwanted antibody isoforms—such as kappa light chain and IgG heavy chain homodimers (150 kDa), and homodimers of the chimeric scFv-heavy-lambda chain (100 kDa)—can form during production and must be removed prior to therapeutic use.
Protein A affinity resins are widely used for antibody capture due to their strong specificity for the Fc domain. However, the eluted material often contains aggregates and unwanted antibody isoforms that must be removed. Strong cation exchange (CEX) chromatography is commonly used as a polishing step to address these impurities. Bispecific antibodies (bsAbs) are ideal test systems for evaluating CEX resins, as they present additional purification challenges such as mispaired chains, charge heterogeneity, lower expression levels, and a higher risk of aggregation.
To characterize the resolution capability of SP PuraBead® Edge, CHO-derived bsAb feedstocks were first processed via a Protein A affinity capture step. The eluate was intentionally stressed—held overnight at pH 9 and then adjusted to pH 5—to increase aggregate content and simulate a real-world purification challenge. Initial separation was performed using a 40 column volume (CV) linear gradient (0–50% elution buffer), followed by a 20 CV gradient to improve run time. Further refinement involved stepwise elution to optimize separation of aggregates and isoforms.
Initial gradient elution
Initial linear gradient elution conditions are captured in Table 1.
Initial results demonstrated promising separation, with four distinct peaks observed, some exhibiting slight overlap and shoulders as expected in a gradient elution (Figure 3A). The SDS-PAGE analysis provided further insight into the separation. Visible in the load sample were various mismatched species, with the bispecific antibody appearing as the most intense band (Figure 3B). Aggregates exceeding 198 kDa were also present, with some material remaining in the well, likely due to size-related diffusion limitations.
(A) Gradient elution separation of four peaks in the bsAbs feedstock
(B) SDS-PAGE analysis of elution fractions
Figure 3: Gradient elution separation of four peaks in the bsAbs feedstock (A) and SDS-PAGE analysis of elution fractions (B).
Each elution peak was analyzed and correlated with the chromatogram, revealing the higher molecular weight species—likely a mismatched homodimer—eluted earlier. This peak aligned with the expected mass of 150 kDa, while the target bispecific antibody, at 125 kDa, predominantly eluted in fraction three. Additional impurities, including high molecular weight species and a potential light chain at approximately 25 kDa, were also detected.
The initial linear gradient conditions show distinct peaks and clear separation potential, providing a strong foundation for further process refinement. Subsequent optimization efforts will focus on improving resolution and reducing run time through gradient and step elution strategies.
Optimized elution
To reduce run time, a 20 CV linear elution (0–50%) of the SP PuraBead® Edge resin was run with the parameters detailed in Table 2.
Parameter
Description
Adsorbent: SP PuraBead® Edge
Column
Equilibration buffer: 50 mM sodium acetate, pH 5.0
Elution buffer: 1 M NaCl, 50 mM sodium acetate, pH 5.6
Load volume: 10 mL (13 mg) of Protein A-purified bispecific feed
Elution gradient: 20 CV elution linear gradient from 0–50% elution buffer
Table 2: Second linear gradient elution conditions, using a 20 CV linear elution, for a high-aggregate bsAb feedstock using SP PuraBead® Edge.
Figure 4 shows the optimized linear gradient elution, which halved the elution run time—from 40 CV to 20 CV—whilst still maintaining resolution of the bsAb and mispaired chains.
Based on the peak resolution observed in the gradient run, a refined step elution strategy was implemented to enhance separation of closely related species. The detailed step elution conditions are provided in Table 3. The stepwise elution strategy enabled complete resolution of six distinct peaks as shown in Figure 5.
Figure 4: Resolution of the bsAb and impurities (mispaired chains and aggregates) resulting from the optimized linear gradient on the SP PuraBead® Edge resin.
Table 3: Step elution conditions for a high-aggregate bsAb feedstock using SP PuraBead® Edge.
Figure 5: Resolution of the bsAb and impurities (mispaired chains and aggregates) resulting from the optimized step elution on the SP PuraBead® Edge resin. Conductivities at which peaks eluted are annotated in black text.
SDS-PAGE analysis of fractions collected at different conductivities provided insight into the separation efficiency and purity of the bsAb (Figure 6). The initial load contained
The fraction collected at a conductivity of 16 mS/cm appears to contain a homodimer or mismatched pair, indicating that these species elute at lower conductivity. The 28 mS/cm fraction contains the majority of the target bispecific antibody, with only minor impurities observed when compared to the load. The 22 mS/cm fraction appears to be of similar molecular weight to the target bsAb but was eluted in its own distinct peak suggesting it could be a possible impurity that was successfully separated. At even higher conductivities, impurity levels increase, leading to some minor loss of the target protein. However, this loss is relatively small compared to the overall yield, suggesting that the separation process is largely effective but may benefit from further refinement to minimize aggregate carryover.
Size exclusion chromatography (SEC) was used to measure the percentage of higher molecular weight species and antibody content in fractions collected from the step elution process. As shown in Figure 7, the load contained about 20% of the higher molecular weight impurities and that was reduced to below 5% in subsequent steps, which can likely be further optimized.
Edge for BsAb aggregate reduction (post-Protein A bispecific feed), reducing aggregates from 20% to <5% using an optimized step elution. Average percentage of higher molecular weight (MW) impurities (yellow) and antibody (teal) content determined by
Optimization of the elution strategy resulted in significant improvements in both resolution and process efficiency. Reducing the linear gradient from 40 to 20 CVs halved the run time, while maintaining effective separation of the bsAb from mispaired species and aggregates. Further refinement using a step elution approach enabled clear resolution of six distinct peaks for more precise fractionation of product-related variants. SDS-PAGE and SEC analysis confirmed improved purity, with high molecular weight impurities reduced from approximately 20% to below 5%. These results demonstrate that SP PuraBead® Edge can be fine-tuned to deliver highresolution polishing.
Conclusion
The results of this study demonstrate the robust performance of SP PuraBead® Edge across key parameters relevant to bsAb purification. The resin maintains high DBC, even under fast-flow conditions, and exhibits strong resolution capabilities across a range of residence times. Compared to competitor resins, SP PuraBead® Edge delivers superior flow characteristics without compromising binding efficiency.
By optimizing elution conditions and leveraging the resin’s high-resolution capabilities, a robust purification process can be achieved, ensuring higher product purity with minimal loss of the target antibody. This includes the effective separation of different isoform species, such as mismatched pairs, light chains, and other impurities, while also reducing aggregate content. These findings highlight the potential of SP PuraBead® Edge as a powerful tool for enhancing bsAb purification workflows, delivering improved efficiency and product quality.
This application note demonstrates that SP PuraBead® Edge is a high-performance, tunable polishing resin, capable of delivering precise, high-resolution separation, even when challenged with high-aggregate conditions and mispaired bsAbs. The ability of the resin to handle complex feedstocks, along with linear and stepwise elution strategies, makes it a strong candidate for platform implementation in bsAb polishing workflows for scalable downstream purification.
Astrea Bioseparations is a world class provider of chromatography adsorbent and resin services. With over 30 years of chromatography manufacturing expertise, we deliver a unique and trusted service in close partnership with our clients. For more information, please don’t hesitate to reach out at sales@astrea-bio.com or visit astreabioseparations.com.