Is Bispecific Antibody Purification Challenging? This Guide Helps You Tackle Complex Impurity Separation!

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In recent years, bispecific antibodies (bsAbs) have remained a major focus in the field of cancer immunotherapy. By the end of 2025, a total of 19 bsAb drugs have been approved worldwide, with more than 300 clinical trials currently underway ^[1]. However, the structural complexity of bsAbs, including light chain mispairing, homodimers, half antibodies, and aggregates, leads to a wide range of product related impurities and poses significant challenges for downstream purification.

As a chromatography resin supplier, BioLink understands that traditional monoclonal antibody purification processes cannot be directly applied to bsAbs. Today, from the perspective of resin selection, we will walk through key considerations for tackling bsAb purification.

Core Challenge in bsAb Purification: Too Many, Too Similar Impurities

Bispecific antibodies can be broadly classified into IgG-like formats with an Fc region and Non-IgG-like formats without an Fc region. For IgG-like bsAbs, especially asymmetric structures such as knobs-into-holes, the co-expression of two different heavy chains and light chains can theoretically generate more than ten types of byproducts, including homodimers, half antibodies, light chain missing species, and aggregates.

What makes purification even more challenging is that these impurities share very similar physicochemical properties with the target molecule, such as pI and hydrophobicity. As a result, conventional ion exchange chromatography often struggles to achieve effective separation.

Resin Selection Strategy: Three-Step Approach with Hydrophobic and Mixed-Mode as Key

Currently, bsAb purification still follows the classic three-step chromatography framework: affinity capture, polishing I, and polishing II. However, the choice of resins needs to be flexibly adjusted based on the structural characteristics of the bsAb.

01 Step 1: Affinity Capture - Protein A Remains the Mainstay

For bsAbs containing an Fc region, Protein A affinity chromatography is still the preferred option for the capture step. Taking MaXtar^®InnovA Pro as an example, it offers a dynamic binding capacity of up to 70 mg hIgG per mL of resin at a 6 minute residence time, and supports in-place cleaning with 0.5 to 1.0 M NaOH, significantly improving process economics. For acid-sensitive antibodies, MaXtar^®HipHA can be selected, enabling efficient antibody elution under mild conditions.

figure-1-chromatogram-of-a-bsab-using-maxtar-hipha-elution-at-ph-4.0.jpg

Figure 1. Chromatogram of a bsAb Using MaXtar^® HipHA, Elution at pH 4.0

Resin Name	Load (mg/mL)	Yield (%)	SEC (%)	rProtein A (ppm)	Elution CV
MaXtar^® HipHA	50	95%	93.5%	3	2.3

Table 1: Chromatogram and Quality Data of MaXtar^® HipHA

01 Step 2–3: Polishing stage - multiple modes show their strengths

For impurities related to complex products, various types of chromatographic media are required. The table below lists common impurity types and recommended chromatographic methods.

Impurity Type	Recommended Resin Type	Mechanism
Aggregates, Fragments	Mixed-mode Cation Exchange	Bind-elute mode, separated by salt concentration gradient
Light chain deletion variants	Mixed-mode Anion/Cation Exchange, Hydrophobic Interaction	Separation based on pI differences (even with small differences)
Half-antibodies, Homodimers	Mixed-mode Cation Exchange	Linear pH gradient elution
Aggregates (Flow-through mode)	Mixed-mode Anion/Cation Exchange, Hydrophobic Interaction	Target product flow-through, impurities binding (mixed-mode anion); Bind-elute mode, separated by salt concentration gradient (mixed-mode cation, hydrophobic interaction)

Table 2: Common impurity types and recommended chromatographic methods

Case 1: Removal of aggregates by hydrophobic interaction chromatography

figure-2-chromatograms-and-quality-data-of-a-bispecific-antibody-on-maxtar-phenyl-hr.jpg

figure-3-chromatograms-and-quality-data-of-a-bispecific-antibody-on-maxtar-phenyl-hr.jpg

Figure 2, 3: Chromatograms and Quality Data of a Bispecific Antibody on MaXtar^® Phenyl HR

MaXtar^®Phenyl HR shows a significant effect in removing aggregates. The sample purity increased from 95.41% to 99.40%, while the aggregate content decreased from 4.59% to 0.60%.

Case 2: Removal of homodimers by hydrophobic interaction chromatography

Figure 4 & Table 3. Chromatographic profile and quality data of a bispecific antibody using MaXtar^® Phenyl HR

MaXtar^® Phenyl HR shows a significant effect in removing homodimers. The sample purity increased from 51.8% to 87.7%, while the hole-hole content decreased from 16.5% to 3.6%.

Case 3: Removal of low-molecular-weight impurities by ion exchange chromatography

Figure 5 & Table 4. Chromatographic profile and quality data of a bispecific antibody using MaXtar^® SP HR

MaXtar^® SP HR shows a significant effect in removing low-molecular-weight (LMW) impurities. The sample purity increased from 92.8% to 99.7%, while LMW content decreased from 7.14% to 0.27%.

Case 4: Removal of acidic and basic isoforms by multimodal chromatography

Sample Name	Yield (%)	SEC (%)	Charge Variants
Sample Name	Yield (%)	SEC (%)	Acid Peak (%)	Main Peak (%)	Basic Peak (%)
Load	/	99.9	45	53	2
Elution	78	100	33	66	1

Figure 6 & Table 5. Chromatographic profile and quality data of a bispecific antibody using MaXtar^® MMC HR

MaXtar^®MMC HR shows a significant effect in removing acidic and basic isoforms. The main peak purity increased from 53% to 66%, while the acidic peak decreased from 45% to 33%.

Summary

The purification of bispecific antibodies is indeed more challenging than that of monoclonal antibodies. However, with appropriate resin selection and process optimization, it is fully possible to achieve high-purity and high-yield separation outcomes.

Reference

[1] PharmaCube NextPharma Database

References

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08 May 2023