In the race to speed up drug development and sharpen the accuracy of treatments, new tools and technologies play pivotal roles. In this article, we hear from several experts who spoke at the 2023 ASMS session on "Accelerating Analysis in the BioPharma Laboratory." Agilent, a key player in this arena, introduces us to a spectrum of technologies facilitating biomolecule analysis. Meanwhile, experts from Janssen Research & Development and MRM Proteomics shed light on pivotal challenges and solutions in the field. Learn how innovations ranging from easy-to-use proteomics kits to 'flash' characterization are shaping the quest to meet urgent healthcare needs.
Innovations in biomolecule analysis
Mike Knierman, the Biopharma Workflow Manager at Agilent, has extensive experience in the field of mass spectrometry and its applications in proteomics and biopharmaceutical analysis. He introduces us to some of Agilent’s key technologies shaping the field of biomolecule analysis. “We have technology encompassing intact and subunits analysis, peptide mapping, released glycans for proteins, impurities and sequence confirmation for oligonucleotides, drug-to-antibody ratio determination, AAV capsid protein profiling, and host cell protein analysis,” says Knierman. “Our workflows are supported by an extensive range of instrumentation for sample preparation, separation, and detection, along with comprehensive software packages for analysis, coordination, and data management.”
One technology Knierman is particularly excited about is ‘flash characterization’ that leverages the microdroplet reaction technique. “By injecting an antibody under native conditions through our systems, one can observe the intact antibody spectrum instantly,” he enthuses. “Adding an IdeS enzyme directly into the autosampler loop and mixing before injection enables us to achieve instant reaction products in the electrospray droplets, significantly speeding up the process by eliminating the need for enzyme incubation.”
Knierman is also keen to highlight Agilent's acquisition of e-MSion, which offers exceptional fragmentation of large molecules with 6545XT AdvanceBio LC/Q-TOF while preserving labile post-translational modifications such as phosphorylation and glycosylation. “This technology enables differentiation of isobaric residues and provides comprehensive sequence information, making it a powerful tool for detailed biomolecule analysis,” he elaborates.
Of course, new technologies require the support of a robust software suite. “Agilent's software stands out for its support of ECD or CID (Collision-Induced Dissociation) fragmentation analysis through the ExDViewer, facilitating real-time streaming and interpretation of fragmentation spectra,” says Knierman. “It allows for on-the-fly adjustments to collision energy, enabling researchers to gain immediate insights into their impact on fragmentation.”
Analytical innovations speed up antibody characterization
Harsha Gunawardena, Principal Scientist at Janssen Research & Development, is a leading expert in the field of proteomics, particularly in the area of rapid and improved characterization of therapeutic antibodies and antibody-related products. He presented the key challenges in antibody development and outlined the solutions that manufacturers such as Agilent are providing.
“Since their emergence in the early 1980s, monoclonal antibodies have been a cornerstone in the field, leading to over 140 therapeutic and diagnostic applications across a spectrum of diseases,” advises Gunawardena. “More recently, the advent of bispecific and multispecific antibodies, capable of targeting multiple antigens simultaneously, has further advanced discoveries. These innovations, particularly CD3 redirectors in cancer therapy, have enabled more precise and potent treatments, marking a new era in antibody-based therapies.”
Gunawardena explains that the development pipeline for antibodies starts with the discovery phase, where numerous antibodies are identified and refined to select promising candidates. This is followed by cell line development, where from a vast pool of clones, one is chosen for advanced development. “Key challenges in this pipeline, especially for bispecific and multispecific antibodies, include managing aggregation, chain mispairing, and post-translational modifications,” notes Gunawardena. “But to ensure a seamless transition from discovery to development, it’s crucial that analytical processes do not become a bottleneck in development.”
He reports that Janssen has addressed these analytical challenges by embracing automation to streamline the end-to-end pipeline. “Our workflow integrates a laboratory information management system (LIMS) for sample management, high-throughput robotics for sample introduction into the mass spectrometer, and cloud-based diagnostic software for rapid data processing.”
A key technique leveraged by Gunawardena and his team is mobile affinity selection chromatography (MASC), which alters traditional column chromatography by incorporating a mobile affinity phase. “This phase has a unique affinity selector reagent that binds selectively to monoclonal antibodies, and it also has a fluorescent tag,” shares Gunawardena. “The process involves the separation of antibody-reagent complexes using a sieving matrix, similar to size-exclusion chromatography, with identification through fluorescence detection.”
MASC has proven to be highly effective at monitoring bioreactor runs. “Using an Agilent 1290 Infinity II LC setup combined with an automated bioreactor system, we've been able to directly identify and quantify monomers, dimers, trimers, and tetramers from the samples without prior purification,” says Gunawardena. “Furthermore, our ability to monitor daily bioreactor runs and analyze supernatants directly has enhanced our workflow efficiency. The Agilent 1290 Infinity II LC's serial injection feature, part of a comprehensive kit including reagents and columns, allowed for efficient sequential sample analysis.”
One more key technology that Gunawardena sheds light on is microdroplet technology. “Microdroplet reactions represent a cutting-edge technique in biochemical analysis, where reactions occur within droplets tens of microns in diameter. This approach significantly accelerates the digestion of antibodies, allowing for rapid characterization. By targeting precise points on antibodies to create subunits, this method enables detailed analysis of post-translational modifications and other critical attributes, making it a powerful tool for exploring the complex nature of antibodies.”
Two main methods generate microdroplets, one involving a mixing tee and the other utilizing emitters to fuse droplets. “Both methods speed up the reaction process by creating optimal conditions for rapid biochemical interactions,” says Gunawardena. He adds that microdroplet technology completes digestion in approximately 250 microseconds, up to 7.5 million-fold faster than bulk reactions. “This enables full digestion into subunits almost instantaneously, demonstrating the technology's potential to streamline antibody characterization workflows.”
This had led to Gunawardena and his team developing systems that allow real-time, online sample introduction to mass spectrometers, reducing cycle times to as little as two minutes. “Utilizing sources such as the Agilent Jet Stream, we generate smaller droplets directed towards the mass spectrometer inlet with high precision, enhancing throughput while maintaining the ability to perform under native pH conditions. We further optimize the process by incorporating autosampler-programmed injections for rapid sample introduction and mixing.”
Advancements in targeted proteome analysis
Christoph Borchers is a Full Professor in the Department of Oncology at McGill University and the Chief Scientific Officer of MRM Proteomics. An expert in the field of multiple reaction monitoring (MRM) mass spectrometry, he has a long-standing collaboration with Agilent to advance the capabilities of quantitative proteomic applications, including structural characterization, tissue imaging, and the development of user-friendly proteomics kits. Borchers shares some of the most recent advances in targeted proteome analysis.
“Our group has advanced MRM techniques, recognized as "Method of the Year" in 2012, to improve the precision of protein quantitation,” says Borchers. “This approach uses stable isotope-labeled peptides identical to those of the proteins being studied, ensuring absolute specificity in measuring protein concentrations. Internal standards are key to this process, enabling precise alignment of precursor and product ions and retention times.”
Borchers adds that MRM technology enables absolute quantitation, allowing the team to determine the exact concentration of proteins in biological fluids and even the copy number in cells. “This advancement is particularly valuable in clinical settings, where precise measurements of protein levels can inform diagnostic and therapeutic decisions. Through the optimization of internal standards, we've achieved sensitivity levels down to a few attomoles on the column.”
A typical service customer request at MRM Proteomics involves the development of assays for specific proteins. With a focus on highly reliable assays, Borchers emphasizes the importance of method validation. “Our approach involves an automated execution of standardized sample preparation protocols—spanning denaturation, reduction, alkylation, quenching, and digestion, along with the incorporation of internal standards and a purification step. Following preparation, samples undergo analysis in an LC/MS system where regression curves and other critical evaluations are performed.”
Speed is another crucial aspect of Borchers’ work. “By integrating the Agilent 6495 LC/TQ with a 1290 UHPLC system, we significantly increased our experiment's throughput, analyzing 375 proteins across three conditions for a total of 2,250 transitions. This required sub-millisecond dwell times in our 60-minute sessions, highlighting the importance of rapid instrumentation.”
The importance of speed and reliability is particularly evident when you consider real-world applications of the technology. “We applied the system using a 270 protein kit integrated with metabolomics to analyze plasma samples from COVID-19 patients in the ICU, aiming to predict outcomes based on the proteins,” reports Borchers. “This analysis serves as a valuable tool for guiding triage decisions in ICUs by predicting patient outcomes with high accuracy from day zero.”
As for future applications, Borchers advises that the research is moving beyond single proteins to study entire pathways, particularly proteins involved in the complement and coagulation pathways. He reveals that the approach is being extended to disease-specific areas, such as cancer, using both plasma samples and cell lines.
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This article is featured in our June 2024 publication, Innovation and Sustainability in Modern Analysis. Find out about the latest innovations and sustainable advances in mass spectrometry, chromatography, and related techniques.
