Orthogonal Analytics: How Much Characterization Is Enough?

Key Takeaways

• Orthogonal analytical methods strengthen biopharmaceutical characterization by providing complementary information about protein structure, aggregation, degradation, and product-related variants that individual techniques may miss.

• Effective analytical method selection depends on understanding each technique’s measurement principles, discriminatory capabilities, and limitations rather than simply increasing the number of assays performed.

• Risk-based characterization connects analytical findings to CQAs, helping biologics developers distinguish molecular differences that affect product quality from variations with limited demonstrated significance.

• Biosimilar comparability and manufacturing change assessments require product-specific analytical strategies, with complementary methods selected to investigate relevant differences and support scientifically justified conclusions.

• Mass spectrometry–based MAMs can support more targeted biologics quality control, potentially replacing established analytical assays when sufficient characterization, validation, and bridging evidence justify the change.

Every additional analytical method executed during biopharma development offers the possibility of revealing something new about a biologic therapeutic. Whether that information improves understanding of product quality is a separate question. As analytical technologies become more capable of resolving subtle molecular differences, developers must determine which measurements are necessary to characterize their products adequately and which add complexity without materially improving the evidence available for development and quality decisions.

The challenge reflects the inherent heterogeneity of biological products. Therapeutic proteins can exhibit differences in higher-order structure, post-translational modifications, aggregation, fragmentation, and other characteristics that may influence their quality, safety, or efficacy. No single analytical technique can adequately characterize every relevant attribute, making complementary approaches essential for establishing a comprehensive understanding of these molecules.

However, extensive product characterization and routine quality control serve different purposes. Characterization establishes knowledge of the molecule, its variants, and its potential vulnerabilities, while specifications define the attributes, analytical procedures, and acceptance criteria used to assess product quality. The International Council for Harmonisation (ICH) recognizes this distinction, emphasizing the selection of quality attributes and methods based on their relevance to the product and associated risks.¹

In biopharmaceutical characterization, orthogonality generally describes the use of analytical techniques that rely on different measurement principles or provide complementary information about a particular product attribute. Two methods may investigate the same characteristic through different physical mechanisms, or they may reveal distinct aspects of a broader quality concern.

Analytical methods have different capabilities and limitations. A technique may resolve certain molecular species while failing to distinguish others, or its measurements may depend on sample preparation, separation conditions, or interactions with the analytical system. A complementary method can provide an independent perspective, helping scientists determine whether an observed result adequately represents the product.

Different technologies, however, do not necessarily provide independent or equally useful information. Two techniques may respond to similar molecular changes despite relying on different measurement principles, while another may reveal characteristics that existing methods cannot detect. Developers must establish complementarity experimentally rather than assuming it from the instruments selected.

In a study published in Frontiers in Chemistry, researchers from the University of Liège in Belgium and the University of Monastir in Tunisia demonstrated these distinctions by comparing three separation techniques for the characterization of adalimumab, a monoclonal antibody (mAb), and etanercept, an Fc-fusion protein, alongside corresponding biosimilars.² The researchers evaluated capillary gel electrophoresis (CGE), reversed-phase liquid chromatography (RPLC), and size-exclusion chromatography (SEC) using untreated samples and samples subjected to stress or reducing conditions.

The techniques provided different information about the products. RPLC separated hydrophilic and hydrophobic degradation products, CGE demonstrated useful selectivity for several adalimumab fragments, and SEC detected aggregates and certain fragments that the other approaches could not adequately resolve. Their relative contributions also depended on the protein under investigation, with CGE proving less informative for etanercept fragments than for adalimumab fragments under the conditions studied.

A method that contributes important information about the degradation behavior of one protein may therefore provide substantially less insight into another. The relevant characterization question and the molecular properties of the therapeutic must inform the selection of complementary techniques.

The importance of method selection becomes particularly apparent when an established analytical technique provides an incomplete representation of the attribute being measured. Protein aggregation and higher-order structural analysis offer examples of how complementary methods can reveal limitations that might otherwise go unrecognized.

SEC has long served as a standard technique for quantifying soluble protein aggregates, but its results depend on the behavior of proteins during chromatographic separation. In a study published in the Journal of Pharmaceutical Sciences, researchers from the University of Colorado, Eli Lilly, and Ludwig Maximilian University of Munich investigated these limitations by comparing SEC with asymmetrical flow field-flow fractionation (AF4) and sedimentation velocity analytical ultracentrifugation (SV-AUC) in acid-stressed and unstressed samples of a recombinant humanized mAb.³

The investigators observed markedly higher aggregate levels with AF4 and SV-AUC than with SEC. The complementary techniques also detected higher-molecular-weight soluble aggregates that SEC failed to identify. Further experiments demonstrated that reinjecting purified antibody dimers onto the SEC column produced detectable quantities of monomers and trimers, while measurements of sample recovery revealed statistically significant antibody losses during chromatography.

The SEC results therefore did not fully represent the aggregate populations present under the experimental conditions. Understanding the behavior of the protein within the analytical system was necessary to interpret the measurements accurately.

Disagreement between orthogonal methods can consequently be informative. Differences in results may reveal limitations in sample recovery, changes induced during analysis, or distinctions in the populations being measured. They do not automatically establish that one technique is correct and another is defective; the measurement principles and experimental conditions must be examined before drawing that conclusion.

Higher-order structural characterization presents a related challenge. Techniques such as circular dichroism (CD), Fourier-transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), fluorescence spectroscopy, and nuclear magnetic resonance (NMR) provide different types of information about protein structure and conformation. Their discriminatory capabilities depend on the nature of the structural differences and the experimental conditions.

In a study published in the Journal of Pharmaceutical Sciences, researchers at Amgen compared several established biophysical techniques with one-dimensional proton profile NMR using mixtures of IgG1 and IgG2 mAbs.⁴ The team found that profile NMR distinguished 93% of the tested sample combinations, compared with 61% for DSC and 52% for near-ultraviolet CD. FTIR and intrinsic fluorescence spectroscopy did not produce statistically significant distinctions among the tested combinations

These results do not establish that NMR is universally superior to other structural techniques, but they demonstrate differences in the ability of the methods to discriminate among particular samples, a capability that must be evaluated in relation to the characterization objective.

The ability to detect increasingly subtle molecular differences raises another challenge: determining which of those differences warrant further investigation or control.

Biological products contain heterogeneous molecular populations, but individual variants do not necessarily carry equivalent implications for product quality. A modification may affect biological activity, stability, or another relevant characteristic, while a different variation may have little demonstrated impact on the product's intended performance.

The concept of critical quality attributes (CQAs) connects analytical characterization with these distinctions. CQAs are properties or characteristics that must be maintained within appropriate limits, ranges, or distributions to ensure the desired product quality. Detailed characterization and accumulated product knowledge support their identification and the development of an appropriate control strategy. The selection of attributes for specification testing is informed by quality risk, together with the suitability of the analytical methods and acceptance criteria used to assess them.^5,6

Antibody glycosylation provides a concrete example of why molecular characterization must be connected to biological function. In a study published in the Journal of Biological Chemistry, researchers at Genentech investigated human IgG1 antibodies lacking fucose in the carbohydrate structures attached to the Fc region.⁷ They found that fucose-deficient antibodies exhibited up to 50-fold greater binding to FcγRIIIa and enhanced antibody-dependent cellular cytotoxicity (ADCC), particularly at lower antibody concentrations. Binding to several other receptors examined in the study was unchanged or showed only modest improvements.

The results establish that a specific glycan modification can have a substantial effect on discrete antibody functions. For a therapeutic whose mechanism of action depends on FcγRIIIa engagement and ADCC, understanding and controlling relevant glycosylation attributes may therefore be especially important. The significance of the same glycan variation must still be assessed in the context of another product's mechanism of action and quality profile.

A more detailed glycan profile may reveal additional species, but their importance depends on their relationship to the therapeutic's intended functions and established quality risks. Where that relationship remains uncertain, new analytical findings may require functional characterization or other investigations before developers can determine whether routine monitoring is warranted.

Additional methods also introduce practical considerations. Analytical procedures require appropriate development, validation, and maintenance when introduced into a control strategy. Each additional routine assay therefore carries analytical performance and lifecycle responsibilities that should be weighed against its contribution to product understanding and control. ICH Q14 provides a framework for science- and risk-based analytical procedure development, while 9 establishes complementary principles for validation.^8,9

Comparability assessments require developers to determine whether differences between products or manufacturing conditions have implications for quality, safety, or efficacy. Two important applications are evaluating a biologic following a manufacturing process change and comparing a proposed biosimilar with its reference product. Although the regulatory contexts differ, both place substantial demands on the quality and interpretation of analytical evidence.

ICH Q5E provides principles for evaluating comparability before and after manufacturing process changes. The guideline emphasizes the collection of relevant technical information to demonstrate that changes have not adversely affected product quality, safety, or efficacy, but it does not prescribe a particular analytical, nonclinical, or clinical strategy for every situation.¹⁰

The necessary characterization depends on the nature of the manufacturing change, the attributes potentially affected, and the existing product knowledge. An additional technique may be justified when established methods cannot adequately resolve an apparent difference or assess an attribute that could have been altered by the process change.

Biosimilar development presents a distinct challenge. Developers must establish similarity to a reference product through comparative evidence, including detailed assessment of relevant physicochemical and functional characteristics. Orthogonal techniques can help distinguish genuine product differences from limitations of individual measurements.

In a review published in BioDrugs, researchers from the United States Pharmacopeia (USP) and AstraZeneca examined the quality attributes and analytical methods used in successful mAb biosimilar applications in the United States and European Union.¹¹ The team analyzed publicly available regulatory review documents containing comparative analytical information for 40 US-approved and 53 EU-approved mAb biosimilars, encompassing 93 review documents associated with approvals through the end of 2024.

The review identified substantial consistency in the core techniques used to evaluate particular quality attributes, alongside variation in supplementary methods. For example, all 40 U.S. and 53 EU applications included peptide mapping for primary-structure assessment. Edman degradation, by contrast, appeared in only nine U.S. and 13 EU applications, where it was used to confirm N-terminal sequence identity or complement peptide mapping to achieve complete sequence coverage.¹¹

The difference illustrates how successful analytical packages can share a common foundation while differing in the supplementary techniques employed. The authors also noted a broader trend toward higher-throughput mass spectrometric methods for sequence confirmation.

These observations demonstrate variation within successful regulatory submissions, although they do not establish that Edman degradation was unnecessary in applications where it was omitted. The inclusion of a method in a successful submission demonstrates its use within an accepted analytical package, but does not establish that the method was indispensable or that a smaller package would have been inadequate.

The review provides a useful picture of established analytical practice without defining a minimum sufficient package. Historical submissions can inform method selection, but product-specific scientific justification remains necessary to determine which complementary measurements are appropriate for a particular comparability assessment.

The relationship between analytical information and decision-making becomes especially important as a biologic approaches commercial manufacturing. During development, extensive characterization helps identify molecular variants, degradation pathways, and attributes requiring control. With increasing product and process understanding, developers can determine which measurements are necessary for routine quality assessment and which are better retained for specialized investigations.

Specifications identify the quality attributes, analytical procedures, and acceptance criteria used to assess product conformity, while the broader control strategy can incorporate process controls and other measures established during development. Comprehensive characterization and commercial specification testing therefore need not employ identical analytical panels.

Traditional profile-based separation techniques, including ion-exchange chromatography (IEC), SEC, and capillary electrophoresis, play important roles in monitoring biologic product heterogeneity. However, their analytical profiles may represent multiple molecular species, including variants with differing implications for product quality. Changes in a chromatographic peak may therefore reveal altered heterogeneity without identifying which modification is responsible or whether the affected attribute is critical.

Mass spectrometry-based multi-attribute methods (MAMs) can provide more direct information about selected molecular modifications within a single analytical procedure. These capabilities create opportunities to replace some profile-based measurements with targeted monitoring of established CQAs, although other techniques may remain necessary to assess attributes that MAM cannot adequately measure.

In a study published in mAbs, researchers at Janssen Pharmaceuticals demonstrated this approach by developing a revised analytical control strategy for a commercial mAb drug product.¹² The team sought to replace an IEC method used to monitor charge heterogeneity with a combination of targeted molecular measurements and complementary purity methods that directly assessed the product attributes identified as critical.

Extensive high-resolution mass spectrometric characterization during development identified numerous post-translational modifications and supported investigations of their relationships to product quality. Stress studies and structure–function analyses established that deamidation in the complementarity-determining region (CDR) was an important acidic charge-variant CQA. Low- and high-molecular-weight species also contributed to relevant changes in the charge profile.

The revised strategy combined a validated liquid chromatography–mass spectrometry (LC-MS) MAM to monitor relevant modifications, including CDR deamidation, with SEC to control high-molecular-weight species and capillary electrophoresis under sodium dodecyl sulfate conditions (CE-SDS) to control low-molecular-weight species. The MAM also incorporated oxidation monitoring and product identity testing.

To support the replacement, the researchers conducted bridging studies connecting the targeted measurements to the established IEC profile. Across 11 drug-product batches stored under different temperature conditions, CDR deamidation measured by MAM showed a strong correlation with acidic variants measured by IEC, with a coefficient of determination (R²) of 0.993. Additional stressed-sample analyses supported the relationship. This correlation, together with the identification of relevant CQAs, complementary purity measurements, and additional bridging studies, supported the scientific justification for replacing IEC rather than establishing direct numerical equivalence between the methods. The authors reported approval of the revised strategy by several health authorities.

The resulting analytical panel monitored the attributes identified as critical without retaining IEC as a separate routine specification method. Importantly, the strategy did not rely on MAM alone. SEC and CE-SDS remained necessary because they addressed relevant molecular populations that the targeted mass spectrometric method did not independently control.

The case demonstrates how extensive molecular characterization can support a more selective routine testing strategy. Establishing which modifications contributed to quality risks enabled the investigators to distinguish necessary measurements from broader profile changes and justify retiring an established assay. The outcome depended on product-specific evidence and cannot be assumed to apply universally to other biologics or testing methods.

This progression reflects the broader life cycle of analytical control strategy development. In a two-part perspective series published in the Journal of Pharmaceutical Sciences in 2025, biotechnology industry scientists and consultants examined the development of integrated analytical control strategies for biologics.¹⁴ The first paper established the foundational principles connecting product characterization, critical quality attributes, and analytical controls,13 while the second presented a stage-appropriate roadmap for implementing those strategies from early development through commercialization.

As product knowledge accumulates, the purpose of analytical testing can shift from broad investigation toward targeted control. New methods may be necessary when they address previously unresolved risks, while established assays can be reconsidered when alternative measurements adequately cover the attributes they were intended to monitor.

An additional analytical method can contribute to characterization in several ways. It may independently confirm an existing result, identify a previously undetected molecular species, or explain discrepancies between established measurements. In each case, developers must determine how the new evidence affects their understanding of the product and the confidence of the intended assessment.

Another possible outcome is that a method provides greater analytical resolution without changing the understanding or management of a relevant quality risk. The additional measurement may distinguish molecular species more clearly, yet provide no evidence that the existing analytical approach fails to address an important attribute.

Deciding whether such a method belongs in the characterization program or routine testing panel requires examining the capabilities of the methods already in use. Developers should establish whether those procedures adequately detect and measure the relevant attributes, whether their known limitations could compromise the intended assessment, and whether the proposed technique offers information that existing measurements cannot provide.

The consequences of any additional findings also matter. A newly detected aggregate population, for example, may reveal a limitation in an existing purity method and warrant further investigation. A structural difference whose functional significance remains uncertain may require further evaluation rather than simply another measurement at greater resolution. A complementary method that independently confirms an established result may strengthen the evidence supporting characterization without necessarily requiring continued routine use.

Analytical sufficiency also depends on the broader control strategy. Direct specification testing is not the only means of addressing a product-quality risk. Established manufacturing controls, process understanding, and validated analytical procedures may collectively support control of relevant attributes, allowing developers to justify excluding an additional routine assay.

A defensible stopping decision therefore requires evidence that the relevant attributes have been identified, the selected methods provide suitable analytical performance, important measurement limitations have been investigated, and remaining uncertainties are adequately addressed by existing product knowledge and controls. The extent of that evidence must reflect the purpose of the assessment and the potential consequences of an unresolved difference.

A method that is valuable for development-stage characterization may not be necessary for commercial release testing, while a technique omitted from routine specifications may become useful during an investigation or following a manufacturing change. Documenting the rationale for method selection and exclusion supports the reassessment of those decisions as the product and its manufacturing process evolve.

ICH Q14 provides principles for analytical procedure lifecycle management, while the stage-appropriate framework described in the two-part perspective series emphasizes the evolution of analytical controls throughout development.

Characterization can be considered sufficient for a particular purpose when the combined analytical evidence and broader control strategy adequately address the relevant quality risks and support the intended decision.

Orthogonal analytical methods provide essential perspectives on the structural and functional characteristics of biologic therapeutics. Their value lies in the distinct information they contribute and the limitations they help address, particularly when existing measurements leave important questions unresolved.

As product knowledge increases, developers can make more discriminating decisions about the methods required for characterization and control. Some uncertainties will warrant additional testing, while others can be adequately addressed through established analytical evidence and manufacturing controls. The commercial MAM experience demonstrates that deeper molecular understanding can even justify retiring established assays. Determining when characterization is sufficient ultimately depends on demonstrating that the chosen methods and broader control strategy adequately address the product's relevant quality risks.

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2. Demelenne, Alice, et al. “Comparison of Three Complementary Analytical Techniques for the Evaluation of the Biosimilar Comparability of a Monoclonal Antibody and an Fc-Fusion Protein.” Frontiers in Chemistry. 9: 782099 (2021).

3. Gabrielson, John P, et al. “Quantitation of Aggregate Levels in a Recombinant Humanized Monoclonal Antibody Formulation by Size-Exclusion Chromatography, Asymmetrical Flow Field Flow Fractionation, and Sedimentation Velocity.” Journal of Pharmaceutical Sciences. 96: 268–279 (2007).

4. Wen, Jie, et al. “A Comparison Between Emerging and Current Biophysical Methods for the Assessment of Higher-Order Structure of Biopharmaceuticals.” Journal of Pharmaceutical Sciences. 109: 247–253 (2020).

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6. Kendrick, Brent S, et al. “Analytical Control Strategy for Biologics. Part I: Foundations.” Journal of Pharmaceutical Sciences. 114: 103826 (2025).

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8. International Council for Harmonisation (ICH). “Q14 Analytical Procedure Development.” U.S. Food and Drug Administration. Mar. 2024.

9. International Council for Harmonisation (ICH). “Q2(R2) Validation of Analytical Procedures.” U.S. Food and Drug Administration. Mar. 2024.

10. International Council for Harmonisation (ICH). “Q5E Comparability of Biotechnological/Biological Products Subject to Changes in Their Manufacturing Process.” U.S. Food and Drug Administration. Jun. 2005.

11. Aithal, Rakesh, et al. “Review of Quality Attributes and Analytical Methods Used for Comparative Analytical Assessment of Monoclonal Antibodies as Part of Successful Biosimilar Approvals in the United States and European Union.” BioDrugs. 40: 713–732 (2026). h

12. Evans, Adam R, et al. “Targeted CQA Analytical Control Strategy for Commercial Antibody Products: Replacing Ion-Exchange Chromatography Methods for Charge Heterogeneity with Multi-Attribute Monitoring.” mAbs. 16: 2341641 (2024).

13. Kendrick, Brent S, et al. “Analytical Control Strategy for Biologics. Part I: Foundations.” Journal of Pharmaceutical Sciences. 114: 103826 (2025).

14. Sampathkumar, Krishnan, et al. “Analytical Control Strategy for Biologics. Part II: Roadmap for Development and Implementation.” Journal of Pharmaceutical Sciences. 114: 103834 (2025).