Biotherapeutics have become one of the fastest growing sectors in the pharmaceutical industry, reaching rates of growth that surpass conventional medicines. While strong efficacy and safety ratings have pushed them into popularity, biotherapeutics require more time to produce due to complex development and testing procedures. Given these products are typically created in living systems, considerable effort must be given to ensuring their correct structure and ultimate purity.
One aspect of biopharmaceutical protein characterization during bioproduction, peptide mapping, has become integral to assessing product quality. Advances in this approach are not only streamlining sample preparation, liquid chromatography (LC), and mass spectrometry (MS) workflows but also supporting expansion of peptide mapping into other uses such as process monitoring.
The importance of peptide mapping
Peptide mapping has become more rapid and convenient as recent advancements in relevant technologies have improved upon the efficiency and reliability of workflows. “Comprehensive protein characterization is crucial for the quality control of biologics, and peptide mapping has become an essential technique for studying the primary structure of proteins. This includes powerful methods that detect and monitor single amino acid changes, modifications, and degradation products,” explains Gurmil Gendeh, Ph.D., marketing manager, pharmaceuticals, at Shimadzu Scientific Instruments. Specific to biologics development, peptide mapping provides initial proof of structure characterization and lot-to-lot identity testing that supports bioprocess development and clinical trials. Given its longevity in biopharmaceutical analysis as a quality control technique, researchers and suppliers alike are constantly looking to improve the process and get more information from the data generated.
The significance of peptide mapping for the assessment of primary sequence and post-translational modifications lies within its ability to be used across several stages in bioproduction, from early development to batch release analysis. “The possibility to employ the same technique with excellent reproducibility and robustness across different analysts and different laboratories spread around the world is critical. With the automation and simplification of sample preparation based rapid digestion protocols using immobilized proteases, and the streamlining of chromatography with new UHPLC systems, such as the Thermo Scientific Vanquish Duo UHPLC, that are capable of doubling throughput on a single system, peptide mapping is a promising tool for batch-to-batch comparison and quality control assessment,” offers Sara Carillo of the NIBRT Institute, “Two peptide maps on one system at the same time. This is a breakthrough for modern QC labs.” Advances in technologies for in-depth analysis at the intact protein level enable peptide mapping to play a crucial role as a confirmatory technique in biotherapeutics development.
Since the technique is used for elucidating the primary structure of large proteins such as antibodies, the complexity of workflows causes some current technologies to suffer reproducibility and sensitivity issues, and difficulty in implementing automated processes. “As a result, considering the huge growth of the development and use of monoclonal antibodies as drugs, the improvement of technologies and methods involved in peptide mapping will be a hot and challenging topic for the biotech and pharmaceutical industry in the next years,” comments Raphael Hopfner, CSO at ProteoGenix.
All in for automation
Full automation of a peptide mapping workflow inline or online with bioprocessing allows a system to perform sample prep, LC separation, and MS independently. Automated processes offer faster sample processing, improved reproducibility, and seamless method transfer from one lab to another. By incorporating technologies like process analytical technologies (PAT) with multi-dimensional liquid chromatography, for example, labs can implement continuous real-time quality assurance for better operational control and compliance.
Shimadzu collaborated with Perfinity Biosciences to develop the Perfinity Workstation, a multi-dimensional automated sample preparation solution. It includes affinity selection, on-column trypsin digestion, desalting and final separation into a mass spectrometer like a quadrupole time-of-flight (Q-TOF) for identification or triple quadrupole LC/MS for quantitation. By automating the workflow in this way, the platform is able to provide a 10-minute cycle time, deliver high reproducibility, and can be applied to different approaches such as multi-attribute methodology (MAM) IgG peptide mapping workflows. It can also be integrated with third-party sampling devices to enable true PAT-like peptide mapping applications.
Reproducibility is a common issue with peptide mapping, presenting variabilities from batch-to-batch and site-to-site. Since not every researcher will produce the exact same protein digest, even if following the same protocols, combining automation into a comprehensive workflow improves the reproducibility of the assay. Agilent has also worked to alleviate this issue by recommending automated preparation and advanced testing materials. Using a robotic liquid handler like the AssayMAP Bravo, that provides a pre-tested sample prep method and can accommodate different digestion protocols, combined with enhanced columns such as Agilent’s AdvanceBio Peptide Mapping and Peptide Plus columns and more sensitive Iterative MS/MS with the 6545XT AdvanceBio LC/Q-TOF, reduces hands-on time and generates higher resolution data.
Further advancing technologies
And while peptide mapping is primarily used for protein characterization, a recent trend is the use of peptide mapping as the foundation for MAM, a tool for simultaneous identification, quantification, and QC. “With the ability to monitor multiple critical quality attributes simultaneously using one assay, MAM is a very efficient and effective quality control tool to potentially replace some of the existing non-MS based assays used for therapeutic protein QC. However, some of the challenges for peptide mapping such as reproducibility, sensitivity, and quantitation capabilities are the same for MAM applications,” explains Lisa Sapp, biopharma segment market manager, at Agilent. Improvements in sample preparation protocols, instrument reproducibility and robustness, and software functions could enable sensible data acquisition and meaningful results from a complex sample run.
Key innovations like MAM for product quality assessment has gained significant interest in the industry, with scientific conferences dedicating full sessions to MAM including regulatory requirements to move the technology into testing and QA/QC. Automated peptide mapping sample preparation platforms such as the Perfinity system will play a key role in adoption of the concept in the bioprocess arena. In addition, advances in mass spectrometry have contributed to streamlined workflows in this area. Tandem mass spectrometry, for example, can fragment peptides into smaller pieces and provide amino acid sequences of the peptides along with any post-translational modifications (PTM), such as glycosylation, phosphorylation, oxidation, and deamidation.
Increases in reproducibility and reduction in sample handling has also been observed with the introduction of magnetic bead technology for high-throughput analysis. “This promises to be a huge step forward in the establishment of a standardized workflow to allow biotherapeutic comparison. In the present scenario where biosimilars are becoming increasingly important within the overall biotherapeutics market, the availability of a robust and reproducible analysis for these complex macromolecules is fundamental to assure molecule stability, efficacy, and safety,” explains Jonathan Bones of the NIBRT Institute. The National Institute for Bioprocessing Research and Training (NIBRT) recently introduced magnetic bead technology into their labs with the Thermo Scientific KingFisher Duo Prime system and the Thermo Scientific Smart Digest Magnetic Kit to speed sample preparation with low hands-on time and increase reproducibility of their peptide mapping workflows.
Hopfner adds that peptide libraries can be a simple and fast way for epitope mapping of monoclonal antibodies and are necessary for MAM methods, accounting for an important step during the biotherapeutic drug development process particularly from a patent and regulatory standpoint. While libraries mostly identify linear epitopes, complex conformational epitopes make current library use challenging.
Earlier this year, a team at the National Institute of Standards and Technology (NIST) released a new CHO peptide library that could provide a resource tool to determine whether detected peptides originate from host cell proteins or the biotherapeutic itself. Created by NIST researchers in collaboration with other institutions including Johns Hopkins University, the library adds to the growing list of peptide libraries available to enhance and speed the peptide mapping process.
Peptide mapping has been around for some time and is definitely here to stay, asserts Carillo. As molecules and medicines continue to become more and more complicated, peptide mapping will continue to play an important part in their overall characterization to facilitate verification of the primary sequence and the identification and quantitation of modification within the sequence.