Figure - uploaded by Pavel V Bondarenko
Content may be subject to copyright.
Source publication
Trypsin digestion can induce artificial modifications such as asparagine deamidation and N-terminal glutamine cyclization on proteins due to the temperature and the alkaline pH buffers used during digestion. The amount of these artificial modifications is directly proportional to the incubation time of protein samples in the reduction/alkylation bu...
Context in source publication
Context 1
... the IgG molecule is relatively difficult to denature due to the rigid CH3 domain in the Fc region [10], making it a good choice for testing the robustness of this trypsin digestion protocol. The amino acid sequence of anti-streptavidin is shown in Table 1 as tryptic fragments. There are 38 heavy chain (HC) tryptic peptides and 15 light chain (LC) tryptic peptides. ...Similar publications
For the past few years, multidimensional liquid chromatography-mass spectrometry (LC-MS) systems have been commonly used to characterize post-translational modifications (PTMs) of therapeutic antibodies (mAbs). In most cases, this is performed by fractionation of charge variants by ion-exchange chromatography and subsequent online LC-MS peptide map...
Peptide mapping by liquid chromatography mass spectrometry (LC-MS) and the related multi-attribute method (MAM) are well-established analytical tools for verification of the primary structure and mapping/quantitation of co- and post-translational modifications (PTMs) or product quality attributes in biopharmaceutical development. Proteolytic digest...
Citations
... The basic structure of therapeutic mAbs can be evaluated using the well-established methodologies of the "bottom-up" protein sequencing approach which involves reduction, alkylation, and proteolytic digestion followed by LC-MS/MS quantification (Ren et al. 2009;Fodor and Zhang 2006). However, quantification of PTMs of degraded states of a therapeutic mAbs using such techniques can be difficult. ...
Mass spectrometry (MS)-based intact mass analysis and structural characterization of biotherapeutic proteins such as monoclonal antibodies (mAbs) are a crucial characterization approach from upstream drug development to downstream product analysis. Due to various endogenous modifications leading to the structural heterogeneity and several N -linked glycan species resulting in macro-heterogeneity, it is challenging to characterize the mAbs. Hence, it is essential to understand the micro-heterogeneity of such proteins with high level of complexity which may vary in charge, size, or hydrophobicity. The development of high-throughput native separation techniques hyphenated with MS with high sensitivity and excellent mass accuracy has improved the top/middle down analysis, intact mass detection, subunit analysis, enhanced sequence coverage, and accurate localization of site-specific modifications. In this review, we have focused on the critical inroads taken for the improvement in MS-based techniques to resolve the challenges related to analysis of mAbs. Various MS-based techniques and their role in high-order structural analysis and the progress in software development have been explained, and further, the challenges remaining have been discussed.
Graphical Abstract
... The digestion is performed with specific proteases. The most commonly used protease for conventional antibodies is trypsin, because it produces peptides in the size range that is most efficient for peptide identification via MS analysis [23,26]. Our MAM platform method uses trypsin as a digestion enzyme [27]. ...
Peptide mapping is an important tool used to confirm that the correct sequence has been expressed for a protein and to evaluate protein post-translational modifications (PTMs) that may arise during the production, processing, or storage of protein drugs. Our new orally administered drug (Ab-1), a single-domain antibody, is highly stable and resistant to proteolysis. Analysis via the commonly used tryptic mapping method did not generate sufficient sequence coverage. Alternative methods were needed to study the Ab-1 drug substance (75 mg/mL) and drug product (3 mg/mL). To meet these analytical needs, we developed two new peptide mapping methods using lysyl endopeptidase (Lys-C) digestion. These newly developed protein digestion protocols do not require desalting/buffer-exchange steps, thereby reducing sample preparation time and improving method robustness. Additionally, the protein digestion is performed under neutral pH with methionine acting as a scavenger to minimize artifacts, such as deamidation and oxidation, which are induced during sample preparation. Further, the method for low-concentration samples performs comparably to the method for high-concentration samples. Both methods provide 100% sequence coverage for Ab-1, and, therefore, enable comprehensive characterization for its product quality attribute (PQA) assessment. Both methods can be used to study other antibody formats.
... The digestion is performed with specific proteases. The most commonly used protease for conventional antibodies is trypsin, because it produces peptides in the size range that is most efficient for peptide identification via MS analysis [23,26]. Our platform MAM uses trypsin as a digestion enzyme [27]. ...
Peptide mapping is an important tool to confirm the correct sequence expressed for a protein and to evaluate protein post-translational modifications (PTMs) that may arise during production, processing, or storage of protein drugs. Our new orally administered drug (Ab-1), a single-domain antibody, is highly stable and resistant to proteolysis. Analysis by the commonly used tryptic map method did not generate sufficient sequence coverage. Alternative methods were needed to study the Ab-1 drug substance (75 mg/mL) and drug product (3 mg/mL). To meet analytical needs, we developed two new peptide map methods with lysyl endopeptidase (Lys-C) digestion. These newly developed protein digestion protocols do not require desalting/buffer-exchange steps, thereby reducing sample preparation time and improving method robustness. Additionally, the protein digestion was performed under neutral pH with methionine acting as a scavenger to minimize artifacts, such as deamidation and oxidation, which are induced during sample preparation. Further, the method for low concentration samples performs comparably to the method for high concentration samples. Both methods provide 100% sequence coverage for Ab-1, and therefore, enable comprehensive characterization for its product quality attributes (PQA) assessment. Both methods can be used to study other antibody modalities.
... Several strategies have been proposed, including the use of additional devices, for example, microwave oven (Pramanik et al., 2002) or proceeding digestion under high pressure (Prinsen et al., 2016;Vilches et al., 2017). These methods were implemented as scientific approaches but they have not been used in routine analysis (Ren et al., 2009). Recently, modified procedures have been proposed to overcome the drawbacks of the standard tryptic protocol and eliminate artificial modifications. ...
The role of mass spectrometry (MS) has become more important in most application domains in recent years. Pharmaceutical analysis is specific due to its stringent regulation procedures, the need for good laboratory/manufacturing practices, and a large number of routine quality control analyses to be carried out. The role of MS is, therefore, very different throughout the whole drug development cycle. While it dominates within the drug discovery and development phase, in routine quality control, the role of MS is minor and indispensable only for selected applications. Moreover, its role is very different in the case of analysis of small molecule pharmaceuticals and biopharmaceuticals. Our review explains the role of current MS in the analysis of both small‐molecule chemical drugs and biopharmaceuticals. Important features of MS‐based technologies being implemented, method requirements, and related challenges are discussed. The differences in analytical procedures for small molecule pharmaceuticals and biopharmaceuticals are pointed out. While a single method or a small set of methods is usually sufficient for quality control in the case of small molecule pharmaceuticals and MS is often not indispensable, a large panel of methods including extensive use of MS must be used for quality control of biopharmaceuticals. Finally, expected development and future trends are outlined.
... Multiple steps in the sample preparation lead to losses, contaminations, biases, and reduced reproducibility (Doellinger et al., 2020), all being important issues in clinical applicability. In addition, by adopting an enzymatic digestion approach, elevated temperature and pH (a part of tryptic digestion) can cause additional artificial modifications, decrease the intensities of "native" peptides and increase the complexity of the sequence identification (Ren et al., 2009). On the other hand, due to the low abundance of endogenous peptides in complex biological matrixes, additional enrichment or fractionation steps are commonly required to separate between low and high molecular-weight molecules. ...
Peptides carry important functions in normal physiological and pathophysiological processes and can serve as clinically useful biomarkers. Given the ability to diffuse passively across endothelial barriers, endogenous peptides can be examined in several body fluids, including among others urine, blood, and cerebrospinal fluid. This review article provides an update on the recently published literature that reports on investigating native peptides in body fluids using mass spectrometry‐based platforms, specifically those studies that focus on the application of peptides as biomarkers to improve clinical management. We emphasize on the critical evaluation of their clinical value, how close they are to implementation, and the associated challenges and potential solutions to facilitate clinical implementation. During the last 5 years, numerous studies have been published, demonstrating the increased interest in mass spectrometry for the assessment of endogenous peptides as potential biomarkers. Importantly, the presence of few successful examples of implementation in patients' management and/or in the context of clinical trials indicates that the peptide biomarker field is evolving. Nevertheless, most studies still report evidence based on small sample size, while validation phases are frequently missing. Therefore, a gap between discovery and implementation still exists.
... To avoid new disulfide bonds forming during sample preparation and analysis, free thiols were capped with N-ethylmaleimide (NEM), and disulfide bond (DSB) scrambling was minimized by digesting at pH 7 with the specific endopeptidase, Lys-C. 38 A short (12 min) reversed phase gradient was developed to provide chromatographic separation of analytes with similar mass to charge ratios that would otherwise be difficult to discriminate by their mass to charge ratio alone (< 2 m/z). An abbreviated liquid chromatography gradient also facilitated automated sample injection, online sample desalting, and rapid column cleaning and re-equilibration. ...
Antibody drug conjugates, a class of biotherapeutic proteins, have been extensively developed in recent years, resulting in new approvals and improved standard of care for cancer patients. Among the numerous strategies of conjugating cytotoxic payloads to monoclonal antibodies, insertion of a cysteine residue achieves a tightly controlled, site-specific drug to antibody ratio. Tailored analytical tools are required to direct the development of processes capable of manufacturing novel antibody scaffolds with the desired product quality. Here, we describe the development of a 12 min, mass-spectrometry-based method capable of monitoring four distinct quality attributes simultaneously: variations in the thiol state of the inserted cysteines, N-linked glycosylation, reduction of interchain disulfide bonds, and polypeptide fragmentation. This method provides new insight into the properties of the antibody intermediate and associated manufacturing processes. Oxidized thiol states are formed within the bioreactor, of which a variant containing an additional disulfide bond was produced and remained relatively constant throughout the fed-batch process; reduced thiol variants were introduced upon harvest. Nearly 20 percent of N-linked glycans contained sialic acid, substantially higher than anticipated for wildtype IgG1. Lastly, previously unreported polypeptide fragmentation sites were identified in the C239i constant domain, and the relationship between fragmentation and glycoform were explored. This work illustrates the utility of applying a high-throughput liquid chromatography-mass spectrometry multi-attribute monitoring method to support the development of engineered antibody scaffolds.
... The former allows the separation of the protein heterogeneity based on the charge properties while the latter allows the identification of mass changes and the exact position of the modification within the protein expressed. However, since peptide mapping requires extensive work and can also generate artefactual modifications, the research for the improvement of this method is ongoing [10,40]. ...
Introduction
In the biopharmaceutical industry, Escherichia coli is one of the preferred expression hosts for large-scale production of therapeutic proteins. Although increasing the product yield is important, product quality is a major factor in this industry because greatest productivity does not always correspond with the highest quality of the produced protein. While some post-translational modifications, such as disulphide bonds, are required to achieve the biologically active conformation, others may have a negative impact on the product’s activity, effectiveness, and/or safety. Therefore, they are classified as product associated impurities, and they represent a crucial quality parameter for regulatory authorities.
Results
In this study, fermentation conditions of two widely employed industrial E. coli strains, BL21 and W3110 are compared for recombinant protein production of a single-chain variable fragment (scFv) in an industrial setting. We found that the BL21 strain produces more soluble scFv than the W3110 strain, even though W3110 produces more recombinant protein in total. A quality assessment on the scFv recovered from the supernatant was then performed. Unexpectedly, even when our scFv is correctly disulphide bonded and cleaved from its signal peptide in both strains, the protein shows charge heterogeneity with up to seven distinguishable variants on cation exchange chromatography. Biophysical characterization confirmed the presence of altered conformations of the two main charged variants.
Conclusions
The findings indicated that BL21 is more productive for this specific scFv than W3110. When assessing product quality, a distinctive profile of the protein was found which was independent of the E. coli strain. This suggests that alterations are present in the recovered product although the exact nature of them could not be determined. This similarity between the two strains’ generated products also serves as a sign of their interchangeability. This study encourages the development of innovative, fast, and inexpensive techniques for the detection of heterogeneity while also provoking a debate about whether intact mass spectrometry-based analysis of the protein of interest is sufficient to detect heterogeneity in a product.
... Individual steps of the sample preparation protocol for the reduced peptide mapping workflow are protein denaturation, reduction of disulfide bonds, optional derivatization of thiol groups using alkylating agents and digestion of the polypeptide with a suitable endoprotease ( Table 2-2). During this procedure, the protein may be exposed to elevated pH and increased temperature, which could induce artificial deamidation, oxidation and hydrolysis of succinimides [23][24][25] and therefore may hinder the ability to accurately determine the abundance of these product quality attributes [5]. To reduce artificial sample degradation, lower pH, lower temperature, and shortened incubation periods are generally preferred, which may in turn compromise the digestion efficiency and could lead to increased levels of peptides generated from missed cleavages and finally may impact the measured product quality attribute abundance. ...
Multi-attribute methods employing mass spectrometry are applied throughout the biopharmaceutical industry for product and process characterization purposes but are not yet widely accepted as a method for batch release and stability testing under good manufacturing practice (GMP) due to limited experience and level of comfort with the technical, compliance and regulatory aspects of its implementation at quality control (QC) laboratories. Here, current literature related to the development and application of the multi-attribute method by peptide mapping liquid chromatography mass spectrometry (MAM) is compiled with the aim of providing guidance for the implementation of MAM in a QC laboratory. This article, focusing on technical considerations, is the first part of a two-tiered publication, whereby the second part will focus on GMP compliance and regulatory aspects. This publication has been prepared by a group of industry experts representing 14 globally acting major biotechnology companies under the umbrella of the European Federation of Pharmaceutical Industries and Associations (EFPIA) Manufacturing & Quality Expert Group (MQEG).
... 41,43,52,53 This is related to the fact that deamidation values can be influenced not only by the sample age but also by the protein structure (primary, secondary, tertiary, and quaternary structures), by environmental factors (pH, hydration, presence of catalysts, etc.), as well as by predepositional human activities (i.e., cooking, excarnation, mummification). 20,42,45,46,54,55 The deamidation value from the same period may vary among publications. 6,16,38,56 This difference can be explained also by the various experimental conditions, from demineralization time to buffer composition and protein extraction procedures, inducing variable deamidation. ...
Peptide mass fingerprinting (PMF) using MALDI-TOF mass spectrometry allows the identification of bone species based on their type I collagen sequence. In the archaeological or paleontological field, PMF is known as zooarchaeology mass spectrometry (ZooMS) and is widely implemented to find markers for most species, including the extinct ones. In addition to the identification of bone species, ZooMS enables dating estimation by measuring the deamidation value of specific peptides. Herein, we report several enhancements to the classical ZooMS technique, which reduces to 10-fold the required bone sample amount (down to the milligram scale) and achieves robust deamidation value calculation in a high-throughput manner. These improvements rely on a 96-well plate samples preparation, a careful optimization of collagen extraction and digestion to avoid spurious post-translational modification production, and PMF at high resolution using matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance (MALDI-FTICR) analysis. This method was applied to the identification of a hundred bones of herbivores from the Middle Paleolithic site of Caours (Somme, France) well dated from the Eemian Last Interglacial climatic optimum. The method gave reliable species identification to bones already identified by their osteomorphology, as well as to more challenging samples consisting of small or burned bone fragments. Deamidation values of bones originating from the same geological layers have a low standard deviation. The method can be applied to archaeological bone remains and offers a robust capacity to identify traditionally unidentifiable bone fragments, thus increasing the number of identified specimens and providing invaluable information in specific contexts.
... Several options to optimize trypsin digestion conditions to minimize modifications on proteins are available. Ren et al. 26 first developed an improved method in 2009 that minimizes trypsin digestion time while maximizing trypsin activity through complete removal of guanidine. Shorter digestion times also reduced trypsin self-digestion and nonspecific cleavages. ...
... Finally, Millan-Martin et al. recently published comprehensive MAM workflows for biotherapeutic characterization and cGMP testing 36 that included detailed protocols of sample preparations used for MAM from previous publications. 9,26 In addition to minimizing sample preparation-introduced artifacts during MAM development, other key aspects should also be assessed, such as digestion efficiency in terms of levels of peptides with missed or nonspecific cleavages. Based on our experience and accumulated knowledge from the literature, to minimize deamidation and oxidation artifacts and maximize the digestion efficiency, we recommend considerations for each step of a typical trypsin digestion protocol (Table 2). ...
The multi-attribute method (MAM), a liquid chromatography-mass spectrometry (LC-MS)-based peptide mapping method, has gained increased interest and applications in the biopharmaceutical industry. MAM can, in one method, provide targeted quantitation of multiple site-specific product quality attributes, as well as new peak detection. In this review, we focus on the scientific and regulatory considerations of using MAM in product quality attribute monitoring and quality control (QC) of therapeutic proteins. We highlight MAM implementation challenges and solutions with several case studies, and provide our perspective on the opportunities to use MS in QC for applications other than standard peptide mapping-based MAM.
