Decoding Complexity: Analytical Workflows for Bispecific Antibodies and Emerging Biologics

The rapid growth of bispecific antibodies and other multispecific biologics has introduced new challenges in analytical characterization, driven by their increased structural complexity and unique quality attributes. This column explores state-of-the-art analytical workflows designed to address these challenges, with a focus on higher order structure analysis, aggregation profiling, and post-translational modification (PTM) characterization. We discuss practical strategies for method development, data interpretation, and control strategy design, alongside an overview of emerging expectations in regulatory filings for these novel modalities. As the field evolves, robust analytical solutions will be essential to ensure product quality, safety, and efficacy.

Bispecific antibodies (bsAb) and multispecific biologics (msAbs) are a large family of molecules that recognize two different epitopes or antigens (1). The dual targeting capacity increases the specificity and therapeutic efficiency in addition to reducing the toxicity associated with traditional mAbs thereby addressing unmet clinical needs (2). Some of the advantages of using bsAb and msAbs over traditional mAbs are mentioned in Table I.

The natural bivalent antibodies are comprised of the two antigen-binding sites that are identical and composed of determinants from both heavy (H) and light (L) chain variable domains, bsAbs, however, can come in many formats. These range from small proteins or non-IgG-like bsAbs that are fragment-based (without an Fc domain) and IgG-like bsAbs. The IgG-like bsAbs generally show good stability and longer half-life in vivo as compared to non-IgG like bsAbs that show enhanced tissue penetration. Another way of classifying the bsAbs is asymmetric or symmetric Fc-bearing molecules with additional domains attached (3).

Fragment-Based Format

The first class combines multiple antigen-binding fragments in one molecule without the Fc region. This arrangement circumvents the chain-association issue, which arises when two different antibody heavy and light chains are co-expressed in one cell line, assuming there is random chain association (Quadroma). A total of 2⁴ = 16 combinations are possible with the desired bispecific antibody yield, making up statistically 12.5% of the total yield (4). The production of these formats is carried out by the co-expression of 1-2 polypeptide chains in lower eukaryotic and prokaryotic expression systems, as the absence of a glycosylated Fc region allows relatively simple production. The Fc-deficient format has lowered costs, but these also have a relatively short plasma half-life that necessitates further reformations to support therapeutic applications (5).

Symmetric Format

The incorporation of both specificities in a single polypeptide chain or HL pair while retaining the Fc region results in a symmetric design. Symmetric formats appear like native antibodies but differ in size and architecture (6). In clinical development, most of the symmetric formats have tetravalent 2+2 designs. One problem with this format is that the close proximity of antigen-binding sites may affect the functional valency, necessitating further optimization in terms of linker length and domain position (7).

Asymmetric Format

The symmetry of H₂L₂ assembly is broken down that requires addressing the chain association issue. The four polypeptide chains are co-expressed to engineer IgG-like bsAbs that consist of different heavy and light chains that are assembled into a chimeric protein that recognizes two different epitopes. It’s interesting to note that bsAbs with regular immunoglobulin G (IgG) architecture (usually) become functionally monovalent for each target (1 + 1) (8). Most asymmetric formats closely resemble natural antibodies by lacking any additional non-native domains or linker sequences that reduce their avidity binding that may affect potency for certain applications (9).

Asymmetric Format and Chain Association Issue

The generation of bispecific heterodimeric IgG antibodies from four antibody chains (two different heavy and two different light chains) in one expression cell line is difficult due to the homo and heterodimerization of heavy chain (HC) and light chain (LC) and is referred to as the chain association issue (10). Sixteen possible H₂L₂ combinations representing ten different antibodies can arise from two different heavy and light chains. The mispairing is of two main types: (i) HC/HC resulting from the homodimerization of heavy chains; and (ii) HC/LC mispairing creates noncognate heavy and light chain combinations (11). Both types of mispairings yield nonfunctional or monospecific molecules whereas the correct asymmetric structure of bsAbs requires that the two different heavy chains form a heterodimer and that each heavy chain pairs with its alternate light chain. The different bsAbs structures necessitate customized purification strategies that support quality and yield requirements (12).

Downstream Purification of bsAbs and msAbs

The bsAbs and msAbs formats can introduce format-specific impurities requiring bespoke analytical assays and purification methods. For example, fragment-based antibodies lack the F_C component, and industry-standard protein-A affinity-based chromatography purification does not apply (13). To increase their half-life, fusion with polyethylene glycol or human serum albumin is done to achieve more favorable PK profiles (14). The general outline for downstream purification of bsAbs and msAbs is shown in Figure 1.

High Throughput (HTP) Production for bsAb Production

The high-throughput (HTP) production of monoclonal antibodies is an important component of an efficient bsAb discovery process as large numbers of molecules are screened thereby increasing the chance of discovering an ideal bsAb.

An ideal bsAb candidate would have desirable antigen binding, biological and molecular functions (15). HTP capabilities would help in removing the multiple rounds of recombinant engineering and screening required to optimize bsAb properties such as potency, selectivity, developability and immunogenicity as shown in Figure 2. The two main issues that delay bsAb production are lowered expression titre and impurities/heterogeneity in purity profiles (16,17). Over the years, newer technologies that are resource and cost effective have been developed that address challenges to developing HTP bsAb production.

High-Throughput (HTP) Production and Characterization of mAb

As the bsAb panels become complex along with combinatorial methods that support the correct pairing of Ab chains, other pre-requisites such as feasibility and complexity associated with bsAb analytics require attention (18). A comprehensive biophysical characterization of post-translational modifications (PTMs) (for example, methionine and tryptophan [Met/trp] oxidation, asparagine [Asn] deamidation, aspartic acid [Asp] isomerization, and Asn-linked glycosylation) along with forced degradation studies can help identify potential issues in early development stages (19). While determining characterization methods for bsAbs, it should be confirmed that selected methods should align with sample requirements for downstream screening assays.

Additionally, heterodimeric antibodies lack the presence of an established purity assay method for quantitative evaluation of heterodimer purity, when potentially several mispaired or undesired species may exist in the expression product. It is expected that the developed analytical method must accurately and reproducibly detect impurities present at 2% or lower level relative to the main desired species. Homodimer impurities can result in different mode of action and potential toxicity or immunogenicity compared to the heterodimeric bispecific antibody. In addition, the homodimerimpurities have a lower stability than the heterodimeric antibody and present a potentially higher risk for aggregation and immunogenicity. Traditional assay methods such as electrophoresis, circular dichroism (CD), differential scanning calorimetry (DSC)- and high-performance liquid chromatography (HPLC)-based methods lack the resolution needed to distinguish these antibody impurities from the desired product (20).

Mass spectrometry (MS) can be used to obtain accurate mass and primary structure information with high accuracy, sensitivity, and selectivity. MS such as electrospray ionization-quadrupole-time of flight (ESI-Q-TOF) can be used to provide isotopic resolution to detect and quantify fully assembled bispecific antibodies and their impurities particularly those arising from modifications such as glycosylation and C-terminal lysine truncation. However, assessment of bispecific antibodies by mass spectrometry gets complicated by heterogeneity arising from N-linked glycans, and heterogeneity because of C-terminal lysine truncation. Reduced MS can confirm bsAb identity; however, aggregate species can interfere with both binding and functional assays, so these should be quantified using analytical chromatography methods (20,21).

Combinatorial approaches for generating bsAb panels such as fragment antigen-binding (Fab)-arm exchange (FAE) produce impurities that show similar properties to the correct bsAb which make their characterization difficult (22). MS helps in navigating this analytical challenge by providing label-free identification and quantification of the heterogenous peptide mixture by linking liquid chromatography methods with electrospray ionization-MS (ESI-MS). The most used method is reversed-phase liquid chromatography-MS (LCMS), which uses an organic phase gradient to separate the sample constituents based on their hydrophobicity under denaturing conditions. This method uses the differences in the hydrophobic profiles of the correctly and incorrectly paired species that allows absolute quantification of each species based on UV absorbance profile (23). For larger bsAb panels or intact IgG format, chromatographic methods need to be complemented with additional orthogonal methods to achieve separation (Table II).

One of the protein engineering approaches is the “knobs-into-holes” (KIH) concept that has been developed to address the bsAb chain association problem. This design strategy involves the mutation of amino acid residues at the interface between CH₃ domains of each heavy chain, where targeted residues are replaced with bulkier amino acids in the “knob” variant and smaller amino acids in the “hole” variant to enforce proper heterodimerization of engineered heavy chains (24).

Antibodies, however, are large molecules (~ 150 kDa), and exhibit structural complexity and heterogenity. KIH engineering can cause changes in the higher order structure of antibodies. The higher-order structure (HOS) of bsAbs refers to the overall three-dimensional (3D) arrangement of the two antigen-binding domains and the linker region that connects them. HOS is critical for the stability, specificity, and functionality of bsAbs, and it can significantly affect their pharmacokinetics and pharmacodynamics (25).

In the case of mAbs, native ion mobility-mass spectrometry (IM-MS) has emerged as a useful structural biology tool to probe the HOS of mAb from just a few micrograms of sample. IM applies charge and rotationally averaged collision cross sections (CCSs) to separate gas phased protein ions on the millisecond timescale. When associated with MS, two ions of the same mass-to-charge ratio but different CCSs can be distinguished. Previously, IM-MS measurements have been shown to monitor the dynamics of bsAb formation resulting from FAE assess antibody-drug conjugate (ADC) structural heterogenity (26).

Recently, a new tool that combines native IM-MS and collision-induced unfolding (CIU), was developed that demonstrated the ability to provide detailed and quantitative data sets on the HOS of bsAbs. Using CIU, protein ions are collisionally heated prior to IM separation to cause protein unfolding in the gas phase. CIU can discriminate against differences based on disulfide patterns, glycosylation levels revealing conformational stability information. The technique that combines CIU with IM-MS provides additional structural information about proteins and protein complexes such as their stability, folding pathways, and subunit interactions along with their size, shape, and charge distribution (27).

Additionally, absolute quantification of IgG-like bsAb species with mispaired LCs can be achieved by MS signal intensity through the spiking of impurities to create a calibration curve. The method uses short (under 5 min) desalting reversed-phase gradients for HTP impurity quantification (28). For analyzing large bsAb panels through combinatorial approaches recently, solid-phase extraction methods have evolved to couple Orbitrap to automated instruments like RapidFire (29). Agilent RapidFire MS (RF-MS) uses a rapid trap and elute strategy to enable desalting and ion sampling coupled to a MS ion source. RF-MS has been shown to allow HTS triage for small molecules and proteins from biochemical buffers in 15 sec as compared to minutes being taken by conventional chromatography (30). The combination of Orbitrap with RapidFire method has demonstrated its ability as a HTP purity screening method for large bsAb panels and shows high adaptability. In the coming years it is envisioned that automated sample preparation and data analysis could allow cell supernatants to bsAb purity data to be around 1000 molecules within a day.

FDA guidance on bispecific antibodies puts more emphasis on the quality, non-clinical and clinical development programs of drugs, to clearly enlist considerations in assessing immunogenicity; and to clarify clinical assessments comparing a bsAbs and an approved monospecific product. FDA recommends that the overall benefit-risk profile of the bispecific antibody should be defined. In many situations, clinical studies for bispecific antibodies will compare the bispecific antibody to standard of care or placebo. FDA guidelines suggest that bsAb characterization should be aligned with standard mAb practices for final regulatory submission (31). This necessitates a more vigorous discovery stage analysis for bsAb versus standard mAb. As such the ability to test larger, more diverse panels at this stage increases the chances of identifying key molecules of interest. In this direction, the ongoing developments to develop automated bsAb workflows and HTP screening assays contribute to streamlining the process to meet the regulatory requirements.

Conclusion

The analytical characterization of bispecific and multispecific antibodies represents a significant leap in complexity compared to traditional monoclonal antibodies, demanding a fundamental shift in our analytical paradigm. As these molecules continue to diversify in format—from fragment-based designs to asymmetric IgG-like structures—each brings unique analytical challenges that cannot be addressed through conventional mAb workflows alone. The chain association issue alone generates multiple product-related impurities with physicochemical properties remarkably similar to the desired product, necessitating sophisticated orthogonal methods combining advanced mass spectrometry, multi-dimensional chromatography, and emerging techniques like collision-induced unfolding. Furthermore, the combinatorial nature of bsAb discovery requires high-throughput analytical capabilities that can screen thousands of variants while maintaining the resolution needed to detect subtle differences in higher-order structure, aggregation profiles, and post-translational modifications.

As regulatory expectations continue to evolve, the field must balance the need for comprehensive characterization with practical constraints of development timelines and resources. Success in this space will ultimately depend on our ability to develop integrated analytical strategies that are both robust enough to ensure product quality and flexible enough to accommodate the remarkable diversity of these next-generation therapeutics. The continued innovation in analytical technologies, from automated workflows to real-time monitoring systems, will be crucial in transforming these complex molecules from promising concepts into safe and effective medicines.

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