The Role of mD-LC–MS for Antibody Analysis in the Biopharmaceutical Industry

The field of therapeutic antibodies has witnessed remarkable growth in recent years, with novel biotherapeutics formats continually emerging. As the complexity of these formats increases, so does the demand for robust analytical methods to characterize them. In this article, we explore the transformative impact of multidimensional liquid chromatography–mass spectrometry (mD-LC–MS) technology on antibody characterization, with a focus on specific performance enhancements. This article explores the challenges faced by traditional chromatographic methods and discusses how mD-LC–MS is changing the way we evaluate high performance liquid chromatography (HPLC) peaks and assess performance. Specifically, this article highlights how mD-LC–MS enables precise peak identification, rapid characterization of complex antibodies, and early detection of post-translational modifications to improve the assessment of potential critical quality attributes (pCQAs) and optimize the overall analytical workflow. This advance has led to significant time savings and a reduction in artificial induced consumption, thereby reducing the risk of sample modifications during analysis.

Before the advent of multidimensional liquid chromatography–mass spectrometry (mD-LC–MS) technology, the development analytics department for therapeutic antibodies at Roche relied solely on various offline chromatographic methods for peak characterization. This involves fractionation of the analytical high performance liquid chromatography (HPLC) method after upscaling on a semi-preparative scale. A sufficient amount of individual peaks or certain areas of the peak pattern are obtained by subsequent preparation of the fractions (pooling, buffer exchange, and concentration). The generated samples are characterized by size-exclusion chromatography (SEC), ion-exchange chromatography (IEC), hydrophobic interaction chromatography (HIC), and reversed-phase (RP) chromatography for peptide mapping analysis. While appropriate for simple peak patterns and standard antibodies, it proved to be a labor-intensive, inefficient, and sample-consuming process. Additionally, the risk of method-induced modifications (such as oxidation and deamidation) during manual fractionation is ever-present. Finally, this methodology is reaching its limit for new and complex antibody formats with a broad range of different species, as observed in IEC chromatograms.

Experimental

Instrumentation and System Configuration

An Agilent InfinityLab 2D-LC System, that has been expanded with additional modules, including pumps and column ovens. In the first dimension, the HPLC method of interest is operated under the same conditions as those used for quality testing. Up to 10 selected peaks for each injection are retained in loops of the multiple heart-cut valve, representing different product species. These cuts are then processed in an automated and sequential manner. In this workflow, analytical C18 RP ultrahigh-pressure liquid chromatography (UHPLC) columns that are identical in dimensions and particles to those used for offline methods were used. We achieved this setup by incorporating short C18 guard columns for peptide trapping after the digestion column (1). This optimization has significantly enhanced the performance of the workflow, enhancing sensitivity, resolution, and peptide sequence coverage.

The introduction of a powerful 2D-LC–MS solution, in conjunction with a script provided by Angi GmbH (Karlsruhe, Germany) that facilitates the capability to integrate multiple chromatographic dimensions, enhanced how the characterization of product variants is performed. The plugin script led to the first proof of concept publication from Gstoettner and colleagues (2) in 2018, enabling subsequent tryptic processing of the obtained fractions. This breakthrough enabled us to develop a versatile mD-LC–MS setup that integrates sample processing (for example, desalting, reduction, enzymatic digestion) and analysis at different molecule levels (intact, reduced, and peptide) within the same chromatographic setup.

By connecting columns and flow paths from various pumps via two-position valves with multiple ports, the system offers high flexibility, allowing for different workflows at the intact, subunit, and peptide levels. To trigger the start times of MS methods and the timetables for additional pumps and valve switching used for the online processing of the cuts, the above mentioned plug-in script (Angi GmbH) enables integration and synchronization of the system components, facilitating automated processing of the cuts within the workflow.

Method Standardization and Implementation

Despite the inherent complexity of mD-LC–MS, the analytical department of Roche’s pharmaceutical development has established standardized and robust methods for routine use in both drug substance and drug product characterization. The transition from manual, offline fractionation to a fully automated online mD-LC–MS workflow has significantly transformed our analytical processes (Figure 1).

FIGURE 1: Workflows for UHPLC peak characterization with mass spectrometry (MS)—offline and online in comparison.

Results and Discussion

The mD-LC–MS workflow described is highly valuable to facilitate the characterization of complex antibody formats. In particular, when a peak identification of a rare sample is needed or when characterization of a product species is time-critical, mD-LC–MS provides precise information about the peaks of interest. Furthermore, it allows us to confidently evaluate and optimize the performance of our UHPLC methods.

Enabling fully automated rapid online analysis mD-LC–MS helps to overcome challenges, which are typically encountered during offline fractionation and conventional characterization workflows. The required upscaling of existing UHPLC methods for the purpose of offline fractionation often negatively impacts separation efficiency, and may lead to impure fractions. In addition, offline fractionation takes significantly longer, and often carries the risk of unintended sample modifications. In retrospect, it becomes difficult to determine the source of these modifications, making it unclear whether these occur during production (upstream or downstream processes) or are an artifact induced by sample preparation steps before the analysis.

Processing Workflows for Antibody Characterization with Mass Spectrometry

In recent years, we have established an array of methods for routine applications to process the fractions obtained online and analyze them using mass spectrometry. These processing methods include intact antibody analysis, reduced antibody analysis, and tryptic peptide mapping (Figure 2).

FIGURE 2: mD-LC–MS sample processing workflows: The first dimension UHPLC is fractionated using multiple heart-cut valves (MHC). Cuts (green) are then processed depending on the analytical question to be identified.Three different routine processing workflows are available: intact antibody analysis, reduced antibody analysis, and tryptic peptide mapping. All workflows are coupled to a mass spectrometer.

Intact Antibody Analysis

Intact antibody analysis involves the direct examination of complete antibodies without enzymatic or reductive cleavage. By preserving the structure, the intact processing method allows information about the integrity of the antibody or potential disulfide bond scrambling between the antibody chains to be obtained. This kind of analysis plays an increasingly important role with ever more complex molecules, for example, bispecific antibody or complex engineered antibody formats.

Reduced Antibody Analysis

Reduced antibody analysis involves reducing disulfide bonds within antibodies, followed by MS analysis. This approach unveils information about antibody subunits, allowing for the precise assignment of clippings to their respective antibody chains. In addition, reduced antibody analysis enables the identification and characterization of various modifications, such as oxidations, pyroglutamate formation, or lysine heterogeneities, specifically attributing them to the individual antibody chains.

Tryptic Peptide Mapping

Peptide mapping processing workflows involve enzymatic digestion (tryptic or LysC) of antibodies into peptides, followed by MS analysis. This method allows for the characterization and relative quantification of modifications at the peptide level, including oxidation, deamidation, isomerization, and others.

The processing method for tryptic peptide mapping on our mD-LC–MS system can be used as an automated sample preparation tool to obtain tryptic peptides, as described in the study by Pot and colleagues (3). Therefore, this method enables efficient and automated PepMap analysis without manual steps such as overnight benchtop digestion of the sample.

As a final point, it is worth mentioning that analysts can switch between the above-listed methods, eliminating the need for manual changing of the system configuration. This unique setup not only simplifies the handling, but the established automation also minimizes the risk of leaks and instrument downtime. Software-driven workflows ensure consistency and reproducibility, empowering researchers to investigate post-translational modifications at top-down, middle-down, and bottom-up level without manual intervention.

Demonstrating Reliability

Within the development departments of Roche/Genentech, themD-LC–MS systems are used across three sites. Given the complexity of the methodologies employed, it is crucial to ensure consistent and reliable analyses across these instruments. These systems must also demonstrate reliability and operational stability.

To validate the reproducibility of the mD-LC–MS methodology, an inter-laboratory comparison of the tryptic peptide mapping workflow was conducted. This inter-laboratory study, published by Camperi and colleagues (4), focused on characterizing the charge variants of Herceptin, a widely used monoclonal therapeutic antibody. The high sequence coverage in each laboratory (ranging from 95% to 97%) enabled consistent analysis of tryptic peptides and meaningful comparison of charge variant levels. The results obtained at all three participating sites showed a high level of agreement, highlighting the reliability, effectiveness, and consistency of the tryptic peptide mapping processing method, which is arguably the most complex workflow currently performed on mD-LC–MS systems.

Furthermore, Grunert and colleagues (5) emphasized the performance and reliability of the mD-LC–MS setup. Our investigation successfully monitored all medium and low-abundant product variants of a bispecific antibody using the online mD-LC–MS approach, demonstrating its value as a complementary and alternative method for analytical method validation. For both above-named studies, the findings are in line with the data obtained through the standard offline procedure, collectively underscoring the consistency and accuracy of this approach.

The Versatility of mD-LC–MS: Key Applications

mD-LC–MS has become useful for several aspects of our work in analytical development for therapeutic antibodies. Some of the key areas where mD-LC–MS has been particularly beneficial include:

Peak Identification and Characterization Within the Assessments of Potential Critical Quality Attributes (pCQA)

As part of regulatory requirements, it is crucial to thoroughly characterize and evaluate the analytical release methods before submission. By streamlining and enhancing efficiency in individual peak characterization of UHPLC methods, mD-LC–MS makes a significant contribution to the in-depth product characterization. The capability of this technology to assess even small individual peaks separately provides high confidence in identifying low abundant pCQAs.

(U)HPLC Method Development

By using mD-LC–MS we can assess the performance of (U)HPLC methods in a relatively straightforward and comparatively fast manner. This capability significantly aids in the development of (U)HPLC release methods. By characterizing chromatograms of different HPLC-based methods at various stages of method development, we can determine the separation efficiency, to ensure a robust quantitative assessment for the most relevant pCQAs in the resulting (U)HPLC method. This knowledge enables us to optimize our analytical methods depending on the specific analytical needs.

Early Detection of Post-Translational Modifications (PTMs)

By integrating the characterization of products by mD-LC–MS in the early stages of the biologics development, most PTMs can be identified with little effort and reduced material consumption. This proactive approach empowers scientists and project teams to swiftly address potential issues related to molecule properties early on, for example, by the adaption of the manufacturing processes or the development of an appropriate control strategy, ensuring patient safety and consistent quality of therapeutic antibodies.

Troubleshooting Activities

Troubleshooting activities, which almost exclusively occur at short notice, are most usually associated with tight timelines and limited sample quantities, thereby requiring an efficient solution. By using mD-LC–MS an unexpected peak detected in an HPLC-based method for a drug substance or drug product can often be characterized rapidly. This allows for a quick assessment of the criticality of the unknown peak, promotes a root cause analysis, and enables the timely implementation of risk mitigation activities.

Process Analytical Technology (PAT) and Process Optimization

mD-LC–MS technology can leverage as a PAT tool for the optimization of critical process parameters that influence product quality for drug product and drug substance. Integrated in close proximity to the bioprocess, it enables real-time monitoring of biopharmaceuticals quality (6). This allows for rapid and fully automated characterization of multiple quality attributes at the peptide level and supports optimizations such as fermentation conditions in upstream- or process parameters in downstream-processing for drug substance. For drug product development, mD-LC–MS is employed to assess the potential impact of different formulation ingredients or sterilization methods on the final product. This helps identify and quantify potential process-induced modifications, ultimately ensuring the product safety and efficacy.

Applications of mD-LC Technology

When examining the immense potential of mD-LC–MS, particularly for analyzing complex antibodies formats, it is clear that it is an increasingly common analytical technique in Roche’s biopharmaceutical development.

mD-LC–MS serves as a powerful tool for in-depth characterization of especially complex molecules. These intricate structures often push traditional analytical methods to their limits, particularly when characterizing the numerous peaks in IEC-based methods. Compared to offline fractionation approaches, mD-LC workflows not only require less sample material, time, and resources, but also surpass the resolution limits of conventional methods. They can enable precise analysis of a molecule’s structure and modifications, addressing critical factors for biopharmaceutical development and quality control and improving product understanding.

Paving the Way for Enhanced Therapeutic Antibodies

While mD-LC–MS has already achieved significant accomplishments for streamlined and automated peak characterization of therapeutic antibodies at Roche, there are areas where further enhancements could make it an even more powerful technique. Developing accessible mD-LC systems, enhancing software features, exploring diverse measures for online sample preparation, and investigating alternative sample processing methods that contribute to addressing specific analytical challenges (7) all serve to refine our analytical capabilities. These advancements not only impact bioprocess development but will also accelerate the delivery of innovative medicines to patients.

Acknowledgments

Many thanks to Michael Leiss and Tobias Graf for reviewing the manuscript.

References

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About the Authors

Katrin Heinrich holds a diploma degree in biotechnology and has been working at Roche Diagnostics GmbH in Penzberg since 2005 as a principal associate scientist.

Saban Özipek holds an M.Sc. in biochemistry and works at Roche in Basel in the analytical department since 2019.

Sina Hölterhoff has been a senior associate scientist in the Biotech Analytics Department at Hoffmann-La Roche since 2018.

Anja Bathke is a senior scientist at Roche in Basel, Switzerland, with more than 15 years’ experience in mass spectrometry analytics and hyphenated techniques of biomacromolecules. .

Martin Winter is a biochemist and works as a principal associate scientist at Roche in Penzberg.

Lucas Hourtoulle has been an associate scientist in the Biotech Analytics Department at Hoffmann-La Roche since 2021.

Tobias Rainer joined Roche Diagnostics Germany in 2023 as a mass spectrometrist in the Extended Characterization department of Analytical Development.