Planning the analytical strategy for product release should be started as early as possible to ensure product quality and regulatory success.
The dual binding capability of bispecific antibodies (BsAbs) can lead to improved safety and efficacy compared to traditional monoclonal antibodies (mAbs). That improvement has led to significant interest in this relatively new class of drugs. As of March 2026, 15 BsAbs had received approval from FDA, most for the treatment of different types of cancer, with the expectation of one for hemophilia A and a second for neovascular age-related macular degeneration and diabetic macular edema.¹ Reports of clinical trial successes with BsAbs also continue to appear on a regular basis,²⁻⁴ which is not surprising given that hundreds of clinical trials for BsAbs are underway.⁵
However, the greater complexity and diversity of BsAbs present development and manufacturing challenges, including for analytical characterization and quality control. BsAbs may or may not contain a fragment crystallizable (Fc) component, and the light and heavy chains can be different.⁵,⁶ Correct chain association is crucial to confirm, but can be difficult to evaluate and requires use of multiple orthogonal methods. In addition to proper heavy and light chain pairing, other key properties must be evaluated, including charge and glycosylation variants, aggregation, fragmentation, stability, binding affinity, purity, and potency, to name a few.⁵,⁷
These analyses are used during development to enable informed decision-making, define appropriate specifications, and support comparability assessments; once validated, they are instrumental in supporting final product release.
The most important analytical methods control identity, purity, and potency, according to Andre Studer, head of bioconjugates analytical development and advanced synthesis with Lonza. The quality control (QC) methods used for product release therefore ensure that key quality attributes such as identity, purity, potency, and safety, meet predefined specifications.
"Because bispecific antibodies and bispecific antibody drug conjugates [ADCs] are structurally diverse, it is important to consider the specific product design, mechanism of action [MOA], and any available data linking these attributes to safety, efficacy, and pharmacokinetics," says Karl Rogerson, director of global analytical development for integrated biologics R&D at Lonza.
Many product-release specifications involve the use of platform or semi-platform methods, according to R. Mark Jones, director of scientific portfolio management at Charles River Laboratories. They include:
For ADCs, Rogerson adds to the list some methods for evaluating the conjugation chemistry (eg, via cysteine, lysine, or glycan-based methods), the levels of free drug, and the drug-to-antibody ratio (DAR). He also points out that ADC payloads are often highly potent and toxic, so even small amounts of free drug can cause serious adverse effects. As such, measurement of free drug levels and using appropriate analytical methods to confirm that they fall within established safety parameters is an essential component of product release QC.
Controlling the DAR is also important, as it determines how many drug molecules are attached to each antibody and directly impacts how effective the drug is, according to Rogerson. He also notes the DAR can influence toxicity, stability, and how the drug is cleared from the body.
Most of these methods typically have well-established workflows that require minimal development and optimization, Jones observes. Mass spectrometry (MS) used alone and in combination with liquid chromatography (LC) [eg, peptide mapping (LC-MS/MS)] is of growing interest⁵,⁶ but still finds limited use in the QC setting. For instance, MS, along with chromatography and electrophoresis, can effectively measure and control homodimer levels, which is particularly important for immune-activating bispecifics, since elevated homodimer levels can increase the risk of cytokine release syndrome.
Potency is the remaining essential analytical method for product release, and it can be complex for BsAbs, particularly BsAbs that are bispecific ADCs. The key, according to Rogerson, is to have a potency method that reflects how the drug works, for example, showing it can bind both targets at the same time using assays like dual-target enzyme-linked immunosorbent assays (ELISAs) or cell-based assays.
Jones notes that, since the opportunity for interactive/interdependent MOA increases as molecular complexity increases, addressing potency may require multiple assays, some of which may be semi-platformized or custom depending upon availability of binding reagents. Cell-based assays, for instance, may include semi-platform methods such as Fc gamma receptor binding or complement activation assays, as well as custom MOA assays requiring extensive cell-line engineering and assay development, he adds.
“While no particular method is of more importance than the other, ensuring efficacy and improved quality of life for patients and their families is of key importance. We need to step away from the trees to see the forest.”
—R. Mark Jones, director of scientific portfolio management at Charles River Laboratories
In addition to the nature of the drug substance, there are human factors that also influence the choice of methods used for product release. Jones emphasized the importance of the discipline and specialty of the subject matter expert. For example, an antibody engineer may consider target binding specificity to be the most important aspect of an antibody drug product, as strong or weak binding can modulate drug efficacy. For a clinician, antibody serum half-life and the protein attributes that modulate this attribute may be the most important aspect of an antibody drug product. The in vitro cell biologist may point to cell-based and MOA-reflecting potency assays, while the stability/formulation scientist will likely focus on the pH and buffer matrix properties.
"While no particular method is of more importance than the other, ensuring efficacy and improved quality of life for patients and their families is of key importance. We need to step away from the trees to see the forest," Jones concludes.
While BsAbs are antibodies, they are more complex and require more extensive analytical testing to ensure they meet product specifications and are of suitable quality for release. In fact, the regulatory approach for bispecific antibodies is still developing, according to Rogerson. The FDA did issue specific guidance for BsAbs in 2021.⁸ Other regulatory bodies generally follow principles similar to those outlined in this document, but with requirements often included within broader biologics guidelines, Rogerson notes.
The challenge relates to the diversity of BsAbs, which come in many different formats, making it difficult to apply a single standard approach. "Consequently," Rogerson says, "developers need to show regulators that they understand how their product behaves and have the right controls in place to ensure it is safe and effective." That may mean, adds Studer, that for some BsAbs and bispecific ADCs, additional analytical evidence beyond what is outlined in the guidance might be required due to structural and functional complexity.
As more BsAbs are approved, however, Jones believes a way for using "default monoclonal antibody practices" that are tailored to the needs and particular quality attributes of each BsAb candidate will be established. "Prior data on adverse events with bispecifics may lead to stronger emphasis on understanding these processes ex-vivo, and unique molecular structures may drive a focus on potential immunogenicity responses, but in the end the regulatory expectations will be driven by strong scientific justification and sponsors' use of data to justify safety as they move through clinical development," he contends.
The best approach for realizing robust, effective analytics for BsAb product release is to lay out the QC analytical strategy during early development phases. Where possible, relying on platform or semi-platform methods for which there is internal (or external, through partners) experience and expertise is also important, according to Jones.
Because potency/MOA analytics are often the most challenging to develop, both Jones and Rogerson emphasize the importance of focusing efforts on these complex methods. "Laying out a potency strategy early on, even if this means recognizing and stating the unknowns, will start a team thinking creatively about the gaps needed to be filled for the long-term strategy," Jones observes.
When developing new methods, Rogerson also highlights the importance of applying the principles outlined in ICH Q14: Analytical Procedure Development.⁹ "This approach aligns with a quality-by-design strategy that starts with defining the analytical target profile, which sets out what needs to be measured and how well the method should perform. Access to this information helps analysts make risk-based decisions, reduce unnecessary lab work, and build strong, reliable methods from the start," he states.
Advances in chromatography and ELISA methods are enabling more rapid QC analysis for BsAbs. With respect to chromatography, Rogerson highlights modern systems that can switch between different columns and mobile phases in a single run, allowing analysts to quickly screen many conditions. Two-dimensional chromatography, meanwhile, can be leveraged to confirm peak identity with mass spectrometry, even when the system does not use MS-compatible buffers, which helps ensure the method is specific and reliable. "These advances have significantly reduced the time needed to develop methods for monitoring levels of homodimers that are fit-for-purpose," he notes.
Studer points to dual-binding ELISAs as an important advance, as they can directly probe simultaneous binding, which is not possible when individual ELISAs are used for each antigen. There is a limitation, however; these assays cannot attribute loss of binding to impairment of a specific binding arm, which can be achieved when 2 individual ELISAs are employed. Often, therefore, these methods are used in combination to achieve comprehensive evaluation of the binding properties of many BsAbs.
The main takeaway for BsAb product release, Rogerson says, is that analytical control for these molecules is mainly about managing their complexity and variability. He again emphasizes the importance of beginning the planning of the analytical strategy as early as possible to ensure product quality and regulatory success. "Depending on the molecule, standard mAb platform methods may not always be suitable. Thinking about the analytical approach early helps make sure the right methods are ready when they are needed," he concludes.
