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Process changes are inevitable in the life cycle of recombinant monoclonal antibody therapeutics. Products made using pre- and post-change processes are required to be comparable as demonstrated by comparability studies to qualify for continuous development and commercial supply. Establishment of comparability is a systematic process of gathering a...
Contexts in source publication
Context 1
... comparability refers to the strategy adopted to ensure that the comparability study is designed to meet phase specific requirements (see Table 2), which vary in depth and scope for different phases of development. 8,9,236,237 Changes during the early phase (prior to Phase 2) are more acceptable due to limited product and process knowledge and clinical validation. ...Context 2
... comparability refers to the strategy adopted to ensure that the comparability study is designed to meet phase specific requirements (see Table 2), which vary in depth and scope for different phases of development. 8,9,236,237 Changes during the early phase (prior to Phase 2) are more acceptable due to limited product and process knowledge and clinical validation. ...Similar publications
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Asparagine deamidation is a common posttranslational modification in which asparagine (Asn) is converted to aspartic acid or isoaspartic acid. By introducing a negative charge, deamidation could potentially impact the binding interface and biological activities of protein therapeutics. We identified a deamidation variant in moxetumomab pasudotox, a...
Purpose
Proteins are the principal biomolecules in bacteria that are affected by the oxidants produced by the phagocytic cells. Most of the protein damage is irreparable though few unfolded proteins and covalently modified amino acids can be repaired by chaperones and repair enzymes respectively. This study reviews the three protein repair enzymes,...
Citations
... One of the most sensitive techniques for the evaluation of mAb charge heterogeneity is capillary iso-electric focusing (cIEF), 8,9 which provides an important tool to investigate the identity, purity, and stability of a mAb and allows product consistency to be monitored during the manufacturing process, covering the whole lifecycle of the product 10 (Figure 1(b)). mAbs contain ionizable acidic and basic groups that can result in a net positive or negative charge depending on the interactions with the surrounding environment. ...
Therapeutic mAbs show a specific “charge fingerprint” that may affect safety and efficacy, and, as such, it is often identified as a critical quality attribute (CQA). Capillary iso-electric focusing (cIEF), commonly used for the evaluation of such CQA, provides an analytical tool to investigate mAb purity and identity across the product lifecycle. Here, we discuss the results of an analysis of a panel of antibody products by conventional and whole-column imaging cIEF systems performed as part of European Pharmacopoeia activities related to development of “horizontal standards” for the quality control of monoclonal antibodies (mAbs). The study aimed at designing and verifying an independent and transversal cIEF procedure for the reliable analysis of mAbs charge variants. Despite the use of comparable experimental conditions, discrepancies in the charge profile and measured isoelectric points emerged between the two cIEF systems. These data suggest that the results are method-dependent rather than absolute, an aspect known to experts in the field and pharmaceutical industry, but not suitably documented in the literature. Critical implications from analytical and regulatory perspectives, are herein thoughtfully discussed, with a special focus on the context of market surveillance and identification of falsified medicines.
... Such assessments are needed to confirm the level of molecular sameness of compounds meant to structurally replicate each other and, to some degree, to predict their pharmacodynamic behavior [1,2]. Comparative analytical assessment can serve to measure the potency of a novel biological medicine, or more commonly, to gauge the risk of unwanted functional consequences of detected minute molecular differences [3]. Functional analytical comparability is therefore increasingly considered a key to the fine-tuned comparison of versions and/or variants of a given biological medicine [4]. ...
... The product-related variants of monoclonal antibodies include size-related variants, charge-related variants, glycosylation variants, post translational modification (PTM) variants, disulfide linkage variants, etc. [7][8][9]. Post-translational modifications (PTMs), chemical modifications, and degradants include frequently observed modifications such as oxidation, deamidation, aspartic acid isomerization, glycation, N/C-terminal modifications, and less common modifications such as hydroxylysine and tyrosine sulfation [10]. As mAbs share a great deal of sequence identity with the exception of the complementarity-determining region (CDR), PTMs that occur in the CDR generally draw more attention during characterization due to their potential to negatively impact potency, immunogenicity, and stability [11,12]. ...
Purpose
Monoclonal antibodies (mAbs), like other protein therapeutics, are prone to various forms of degradation, some of which are difficult to distinguish from the native form yet may alter potency. A generalizable LC–MS approach was developed to enable quantitative analysis of isoAsp. In-depth understanding of product quality attributes (PQAs) enables optimization of the manufacturing process, better formulation selection, and decreases risk associated with product handling in the clinic or during shipment.
Methods
Reversed-phase chromatographic peak splitting was observed when a mAb was exposed to elevated temperatures. Multiple LC–MS based methods were applied to identify the reason for peak splitting. The approach involved the use of complementary HPLC columns, multiple enzymatic digestions and different MS/MS ion dissociation methods. In addition, mAb potency was measured by enzyme-linked immunosorbent assay (ELISA).
Results
The split peaks had identical masses, and the root cause of the peak splitting was identified as isomerization of an aspartic acid located in the complementarity-determining region (CDR) of the light chain. And the early eluting and late eluting peaks were collected and performed enzymatic digestion to confirm the isoAsp enrichment in the early eluting peak. In addition, decreased potency was observed in the same heat-stressed sample, and the increased isoAsp levels in the CDR correlate well with a decrease of potency.
Conclusion
Liquid chromatography-mass spectrometry (LC–MS) has been utilized extensively to assess PQAs of biological therapeutics. In this study, a generalizable LC–MS-based approach was developed to enable identification and quantitation of the isoAsp-containing peptides.
... Despite the extensive structural diversity observed, the top ten glycans found in all groups share eight common terminal epitopes, which are highlighted on glycan structures in Table 1. These eight epitopes consist of non-immunogenic ones: core fucose, terminal N-acetylglucosamine (GlcNAc), terminal galactose, high mannose, triantennary structure, and terminal NANA with an exclusive α2,3-linkage on IgG1 monoclonal antibodies produced in CHO cells [24]. Additionally, there are two potentially immunogenic epitopes, namely α-galactose and terminal NGNA with mostly α2,6linkage [25][26][27]. ...
Purpose
This study aims to establish a benchmark glycan profile for commercial therapeutic monoclonal antibodies (mAbs) approved by the US Food and Drug Administration (FDA).
Methods
We conducted a rigorous comparison of glycosylation data from the regulatory submissions for FDA-approved therapeutic antibodies up to May 2023. This analysis includes over 150 mAbs produced by various mammalian cell expression systems.
Results
The study identified nine prevalent glycan epitopes across all FDA-approved monoclonal antibodies produced by different expression systems. These epitopes include terminal N-acetylglucosamine, core fucose, terminal galactose, high mannose, α-galactose, terminal α2,3-linked N-acetylneuraminic acid, terminal α2,6-linked N-glycolylneuraminic acid, triantennary structure, and bisecting N-acetylglucosamine, thus establishing a benchmark glycan profile.
Conclusions
The findings of this study have significant implications for therapeutic antibody development, quality control, and regulatory compliance. The benchmark glycan profile enables the assessment of glycosylation consistency and comparability across a diverse range of antibody products, ensuring improved product quality within the biopharmaceutical industry.
... The HOS of therapeutic mAbs is assessed with multiple biophysical techniques during process and product development to ensure that structural integrity, product quality, safety, and efficacy are maintained in the manufactured drug substance from batch to batch. There are several mainstream biophysical techniques that can be used to monitor HOS during biotherapeutic development such as far-UV and near-UV circular dichroism (CD), Fourier transform infrared (FTIR) spectroscopy, fluorescence spectroscopy, and differential scanning calorimetry (DSC) [7][8][9][10][11][12]. Cryogenic electron microscopy (cryo-EM), X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, hydrogen/deuterium exchange mass spectrometry (HDX/MS), and fast photochemical oxidation of proteins-mass spectrometry (FPOP-MS) are higher resolution techniques that currently require subject matter experts to acquire, process, and interpret the data generated in a biopharmaceutical lab [13][14][15][16][17][18]. ...
Purpose
Nuclear magnetic resonance (NMR) spectroscopy provides the sensitivity and specificity to probe the higher order structure (HOS) of monoclonal antibodies (mAbs) for potential changes. This study demonstrates an application of chemometric tools to measure differences in the NMR spectra of mAbs after forced degradation relative to the respective unstressed starting materials.
Methods
Samples of adalimumab (Humira, ADL-REF) and trastuzumab (Herceptin, TRA-REF) were incubated in three buffer-pH conditions at 40°C for 4 weeks to compare to a control sample that was left unstressed. Replicate 1D ¹H and 2D ¹H-¹³C HMQC NMR spectra were collected on all samples. Chemometric analyses such as Easy Comparability of HOS (ECHOS), PROtein FIngerprinting by Lineshape Enhancement (PROFILE), and Principal Component Analysis (PCA) were applied to capture and quantitate differences between the spectra.
Results
Visual and statistical inspection of the 2D ¹H-¹³C HMQC spectra of adalimumab and trastuzumab after forced degradation conditions shows no changes in the spectra relative to the unstressed material. Chemometric analysis of the 1D ¹H NMR spectra shows only minor changes in the spectra of adalimumab after forced degradation, but significant differences in trastuzumab.
Conclusion
The chemometric analyses support the lack of statistical differences in the structure of pH-thermal stressed adalimumab, however, it reveals conformational changes or chemical modifications in trastuzumab after forced degradation. Application of chemometrics in comparative NMR studies enables HOS characterization and showcases the sensitivity and specificity in detecting differences in the spectra of mAbs after pH-thermal forced degradation with respect to local and global protein structure.
... Different levels of information are obtained from various studies including analytical studies for characterization of the molecule, animal studies for assessing toxicity, pharmacokinetics and pharmacodynamics, and clinical studies for assessing safety/tolerability, immunogenicity and efficacy. Analytical characterization of protein structure and function forms the foundation of comparability demonstration [8][9][10][11] which can independently avoid conducting expensive and time-consuming non-clinical and clinical studies [12]. Cutting-edge multi-level analytical and characterization tools for mAbs and ADCs to demonstrate comparability are well established in the literature [13][14][15][16][17], and forced-degradation studies further aid in teasing apart significant quality differences when performed side-by-side [18]. ...
ARX788 is an anti-HER2 antibody drug conjugate (ADC) developed using Ambrx proprietary Engineered Precision Biologics technology. The manufacturing process of ARX788 has been optimized during the course of early to late-phase clinical development. A comprehensive evaluation of side-by-side comparability between pre- and post-change process for ARX788 drug substance and drug product from a quality perspective was conducted based on ICH Q5E guidelines consisting of batch release assays, physicochemical and biophysical characterization, biological characterization, and forced degradation studies. All results have substantiated a high degree of similarity between the pre- and post-change ARX788 drug substance batches and drug product lots, demonstrating that the process manufacturing changes did not impact product quality.
... Since C1q interacts with the CH2 domain in the Fc part of the antibody, the PTMs forming here may affect the C1q interaction [47]. Studies report that Met252 oxidation in the Fc part reduces C1q binding affinity [48]. ...
Alterations in the biological activity of the molecules under stress conditions have not been documented as widely in the literature yet. This study was designed to reveal the functional impacts of various oxidation conditions on a model mAb, a commercial anti-VEGF IgG molecule. The responses to antigen binding, cell proliferation, FcRn receptors, and C1q binding, which rarely appear in the current literature, were investigated. The authors report peptide mapping data, post-translational modification (PTM) analysis, cell proliferation performance, and antigen (VEGF), C1q, and FcRn binding activities of the mAb under various stress conditions. The oxidation-prone site of the mAb was determined as Met252 in the DTLMISR peptide. The VEGF binding activity and anti-cell proliferation activity of the mAbs did not alter, while C1q and FcRn binding capacity significantly decreased under oxidative stress conditions. The full report is vital for many scientific and industrial processes about mAbs. The authors recommend performing functional analyses in addition to the structural studies while investigating the impacts of stress factors on therapeutic mAbs.
... Oligonucleotide mapping provides a direct, detailed assessment of mRNA primary structure comparability across multiple mRNA DS batches. This is analogous to application of peptide mapping by LC-UV-MS/MS for comparability assessment of therapeutic protein batches 26 . The mRNA primary structure of three commercial BNT162b2 batches were deemed comparable ( Fig. 2A), as demonstrated by the superimposition of the full-length chromatograms and by the superimposition of zoomed segments of the chromatograms (Supplementary Data Fig. 3). ...
Oligonucleotide mapping via liquid chromatography with UV detection coupled to tandem mass spectrometry (LC-UV-MS/MS) was recently developed to support development of Comirnaty, the world’s first commercial mRNA vaccine which immunizes against the SARS-CoV-2 virus. Analogous to peptide mapping of therapeutic protein modalities, oligonucleotide mapping described here provides direct primary structure characterization of mRNA, through enzymatic digestion, accurate mass determinations, and optimized collisionally-induced fragmentation. Sample preparation for oligonucleotide mapping is a rapid, one-pot, one-enzyme digestion. The digest is analyzed via LC-MS/MS with an extended gradient and resulting data analysis employs semi-automated software. In a single method, oligonucleotide mapping readouts include a highly reproducible and completely annotated UV chromatogram with 100% maximum sequence coverage, and a microheterogeneity assessment of 5′ terminus capping and 3′ terminus poly(A)-tail length. Oligonucleotide mapping was pivotal to ensure the quality, safety, and efficacy of mRNA vaccines by providing: confirmation of construct identity and primary structure and assessment of product comparability following manufacturing process changes. More broadly, this technique may be used to directly interrogate the primary structure of RNA molecules in general.
... Nonenzymatic fragmentation has been reported to be catalyzed by interaction with copper and iron; is highly pHdependent; and is typically observed around the hinge region, domain−domain interface, and the complementary-determining region. 35,36 Fragmentation of a mAb, however common, is usually not a major concern due to the drug product being highly purified and formulated in conditions which protect from fragmentation (or other degradation pathways). Interestingly, characterization of the C239i mAb intermediates by reduced capillary gel electrophoresis (CGE) revealed a unique and unexpected fragmentation pattern ( Figure 5A). ...
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.
... Different levels of information are obtained from various studies including analytical studies for characterization of the molecule, animal studies for assessing toxicity, pharmacokinetics and pharmacodynamics, and clinical studies for assessing safety/tolerability, immunogenicity and efficacy. Analytical characterization of protein structure and function forms the foundation of comparability demonstration which can independently avoid conducting expensive and time-consuming animal and clinical studies [8][9][10][11]. ...
ARX788 is an anti-HER2 antibody drug conjugate (ADC) developed using Ambrx proprietary Engineered Precision Biologics technology. The manufacturing process of ARX788 has been optimized during the course of early to late-phase clinical development. A comprehensive evaluation of side-by-side comparability between pre- and post-change process for ARX788 drug substance and drug product from a quality perspective was conducted based on ICH Q5E guidelines consisting of batch release assays, physicochemical and biophysical characterization, biological characterization, and forced degradation studies. All results have demonstrated a high degree of similarity between the pre- and post-change ARX788 drug substance batches and drug product lots, demonstrating that the process manufacturing changes did not impact product quality.
