As therapeutics, monoclonal antibodies are remarkable. They are highly specific for their molecular targets. They have slow clearance rates and can provide intended therapeutic effects for weeks following administration. And they can be directed against potentially deadly and difficult-to-treat conditions, providing longer and better lives for patients affected by diseases such as breast cancer and rheumatoid arthritis.
Like any drug, monoclonal antibodies must be extensively characterized throughout development and manufacturing to meet regulatory requirements and to ensure product safety and efficacy. However, as 150 kDa multi-subunit proteins, mAbs are large and structurally complex. They are also highly heterogeneous. Thousands of variant combinations can exist for a given mAb.1 The need to extensively characterize mAbs at the molecular level therefore presents a unique challenge to drug developers and manufacturers. The pressing need to meet this challenge has resulted in the creation and ongoing refinement of imaginative laboratory methods.
While mass spectrometry has emerged as the gold standard, it is not the only means available for the structural characterization of mAbs. The translation of Western assays from the old slab gel format to automated capillary electrophoresis-based separation and immunodetection assays, such as those accomplished by ProteinSimple’s Simple Western platforms, enables productive application of Westerns to mAb clone selection, determination of antibody stability, and other broad molecular features. “We have customers who use mass spec and our Simple Western assays orthogonally,” says Andrea Tu, marketing applications manager at ProteinSimple. They’ll use Simple Westerns up-front, as a pre-screen, or after their mass spec work as a validation. Simple Western also helps people meet their regulatory requirements for submitting multiple methods of characterization,” adds Dr. Tu.
While mass spectrometry has emerged as the gold standard, it is not the only means available for the structural characterization of mAbs.
In liquid chromatography, orthogonal separations methods have been made more powerful by improved first-to-second dimension interfaces that preserve first-dimension separations at high resolution and allow “heart-cutting” of select peaks for small-volume injection. Aided by these improvements, certain mAb modifications, such as glycosylation, can be detected without MS.
For detailed characterization of mAbs, though, advancements in complex protein mixture separation technologies are more likely to be applied to the front-end requirements of mass spectrometry, which works best with purified, simplified, well-separated samples. With the ongoing development of increasingly powerful mass spectrometry instrumentation, the sample-preparation requirements are changing, and new avenues of mAb characterization are becoming available.
Top-down analysis
It appears increasingly inevitable that top-down proteomics will eventually become a routine, practical approach to mAb characterization. Demonstrating the relative insensitivity of top-down MS to sample complexity, He, et al., recently described the use of a 21 tesla FT-ICR MS/MS system to achieve extensive cleavages of both variable and constant antibody regions and 53% sequence coverage from their analysis of mAbs in a complex human serum mixture.2 Though their customized instrumentation system and unique approach precludes broad implementation, their study demonstrates the potential value of the top-down approach and encourages further exploration and development.
Citing advantages such as easier sample preparation, fewer artifacts, and simpler analysis, top-down enthusiasts Bondarenko, et al., demonstrated mass measurement of intact mAbs, and resolution of different glycoforms, using a Thermo Fisher LTQ Orbitrap mass spectrometer and Ion Max ESI/API source operated at elevated voltages and temperature.3 More recently, at the ASMS Asilomar Conference on Nation Mass-Spectrometry-Based Structural Biology, held in October 2015, representatives from Thermo Scientific and Bruker Daltonics updated attendees on commercial development of spectrometry instrumentation for large molecule analysis. Revealing strong user interest in improved methods for top-down mAb characterization, a representative of LC-MS service provider MS Vision described a hybrid system composed of a Thermo Velos source coupled to a Waters QTof instrument.
Accordingly, in February of 2017, The Consortium for Top-Down Proteomics, a non-profit organization dedicated to intact protein analysis, announced the initiation of a multiple laboratory study for characterizing mAbs by top-down MS techniques. The group presented an initial report at the June 2017 ASMS Annual Conference.
Middle-up analysis
Practitioners of middle-up mAb characterization take advantage of the superb capabilities of newer mass spectrometry systems in what is now the middle-mass range. Recent developments in protein digestion options and chromatographic separations have contributed to the popularity of the middle-up approach.
In middle-up analysis, the mAb heavy chains are cleaved at the hinge region. The resulting products are then reduced to produce six 25 kDa fragments. Previous protocols called for the use of pepsin, Lys-C, or papain to effect this cleavage,4 but the difficulty in obtaining accurate, reproducible substrate recognition and enzymatic activity at the hinge region limited the accessibility of the middle-up method.5 Recently, the IdeS (Streptococcus pyogenex) enzyme has been found to perform the desired cleavage selectively and reliably, greatly improving the practicality of the middle-up approach.6 Combined with innovative chromatographic separation techniques. IdeS digestion has enabled domain-specific characterization of reduced mAb fragments by middle-up mass spectrometry.
In a study published in 2017, D’Atri et al., used the fragmentation strategy described above and hydrophilic interaction chromatography (HILIC) to resolve glycosylation-related hydrophilic variations between originator and biosimilar mAbs by middle-up mass spectrometry.7 The authors noted that the recent availability of wide-pore HILIC phases, such as the Waters Acquity UPLC Glycoprotein Amide 300 Å used in the study, recently made this MS-compatible separation applicable to large biomolecules and middle-up mAb characterization studies.
Biocompare’s Mass Spec Search Tool
Find, compare and review mass spec
systems from different suppliers Search
A 2017 study by Leblanc, et al.,8 describes the use of ion exchange chromatography with MS to accomplish middle-up characterization of IdeS-digested mAb. This study is of particular interest because conventional ion exchange mobile phases are not compatible with mass spectrometry, and an offline-desalting step, which slows workflow and degrades chromatographic resolution, is usually a requirement for IEX-MS. However, the study describes successful use of the volatile salts ammonium formate and ammonium acetate with the ThermoScientific MabPac SCX-10 ion exchange column coupled to native mass spectrometry. By programming a mixed-mode IEX separation based on both pH and salt gradients, the study accomplished relative quantification of different isoforms present in a mAb that had been aged for five years.
Bottom-up analysis
The continuing development, improvement, and application of top-down and middle-up mass spectrometry strategies of mAb characterization may be viewed by some as part of an effort to reduce exposure to the limitations and complexities of peptide mapping as accomplished by bottom-up mass spectrometry. However, peptide mapping is an essential technique for mAb characterization that provides primary sequence information as well as the identification, locations, and quantitation of mAb post-translational modifications. IdeS-enabled middle-up analysis can provide simultaneous characterization of mAb subunit microheterogeneities, but peptide mapping is still required in many cases for the determination of the exact cause of differences between two samples. Therefore, bottom-up mAb analysis is still an important target for improvement.
Liquid-handling automation is now taking a role in the development of better bottom-up workflows by minimizing labor-intensive and error-prone manual sample preparation. According to Maryann Shen, automation solutions marketing manager at Agilent Technologies, the company is using AssayMAP Bravo technology to ensure consistent results in the bottom-up characterization of mAbs. The AssayMAP is able to provide capabilities for desalting and digestions using microchromatography separation implemented in a 96-well plate liquid handling platform. This results in higher throughput (processing 1–96 samples at a time), faster digestion through desalting, and superior reproducibility through quantitative binding and high sample recovery. The AssayMAP can also perform protein A, protein G or streptavidin affinity purification of mAbs in complex matrices prior to digestion, adds Shen.
Conclusions
For scientists involved in the development and manufacture of mAbs, there is no single method or workflow that can claim to handle all mAb characterization requirements. An overall approach that anticipates the need for complementary methods and technologies is a necessity. Mass spectrometry sits firmly at the center of the majority of productive mAb characterization methods, and of all the ingenious applications of MS to mAb characterization, bottom-up peptide mapping is the most essential. The middle-up approach, now with the reliable subunit generation afforded by the IdeS enzyme, and its potent utilization of high mass range MS instrumentation is increasing in importance and deserves present consideration.
References
1. Koen, S, et al., “Characterizing Monoclonal Antibodies and Antibody–Drug Conjugates Using 2D-LC–MS,” LCGC Europe 30(3):149–157, 2017
2. He, L, et al., “Analysis of Monoclonal Antibodies in Human Serum as a Model for Clinical Monoclonal Gammopathy by Use of 21 Tesla FT-ICR Top-Down and Middle-Down MS/MS,” J Am Soc Mass Spectrom, 28(5):827-838, 2017. [PMID: 28247297]
3. Bondarenko, PV, et al., "Mass measurement and top-down HPLC/MS analysis of intact monoclonal antibodies on a hybrid linear quadrupole ion trap-Orbitrap mass spectrometer," J Am Soc Mass Spectrom, 20(8):1415-24, 2009 [PMID: 19409810]
4. Fekete, S, et al., “Analytical strategies for the characterization of therapeutic monoclonal antibodies,” Trends Anal. Chem, 42:74-83, 2013
5. Yunyu, L, Zang, Li, “Subunit Analysis Leads to Simultaneous Characterization of Multiple Attributes of Monoclonal Antibodies,” American Pharmaceutical Review
6. Yan, A, et al., “A new tool for monoclonal antibody analysis: Application of ideS proteolysis in IgG domain-specific characterization,” MAbs, 6(4):879-93, 2014 [PMID: 24927271]
7. D’Atri, V, et al., “Hydrophilic Interaction Chromatography (HILIC) hyphenated with Mass Spectrometry: a powerful analytical tool for the comparison of originator and biosimilar therapeutic monoclonal antibodies at the middle-up level of analysis,” Anal Chem, 89(3):2086-2092, 2017 [PMID: 28208257]
8. Leblanc, Y, et al., “Charge variants characterization of a monoclonal antibody by ion exchange chromatography coupled on-line to native mass spectrometry: Case study after a long-term storage at +5 °C,” J Chromatogr B Analyt Technol Biomed Life Sci, 2017 Mar 24;1048:130-139 [PMID: 28242492]
Image: Shutterstock Images