Why is there no biosimilar of Erbitux®?

Monoclonal antibody (mAb)-based therapies have been a major advance in oncology patient care, even though they represent a significant healthcare cost. Biosimilars, launched in Europe in 2004 are an economically attractive alternative to expensive originator biological drugs. They also increase the competitiveness of pharmaceutical development. This article focuses on the case of Erbitux® (cetuximab). This anti-EGFR (Epidermal Growth Factor Receptor) monoclonal antibody is indicated for metastatic colorectal cancer (2004) and squamous cell carcinoma of the head and neck (2006). However, despite the expiration of the patent in Europe in 2014 and estimated annual sales of 1.681 million US dollars in 2022, Erbitux® has not yet faced any approved biosimilar challenges in the United States or in Europe. Here, we outline the unique structural complexity of this antibody highlighted by advanced orthogonal analytical characterization strategies resulting in risks to demonstrate biosimilarity, which may explain the lack of Erbitux® biosimilars in the European and US markets to date. The development of Erbitux® biobetters are also discussed as alternative strategies to biosimilars. These biologics offer expected additional safety and potency benefits over the reference product but require a full pharmaceutical and clinical development as for New Molecular Entities.

Effects of the COVID-19 pandemic: new approaches for accelerated delivery of gene to first-in-human CMC data for recombinant proteins.

The COVID-19 pandemic highlighted the urgent need for life-saving treatments, including vaccines, drugs, and therapeutic antibodies, delivered at unprecedented speed. During this period, recombinant antibody research and development cycle times were substantially shortened without compromising quality and safety, thanks to prior knowledge of Chemistry, Manufacturing and Controls (CMC) and integration of new acceleration concepts discussed below. Early product knowledge, selection of a parental cell line with appropriate characteristics, and the application of efficient approaches for generating manufacturing cell lines and manufacturing drug substance from non-clonal cells for preclinical and first-in-human studies are key elements for success. Prioritization of established manufacturing and analytical platforms, implementation of advanced analytical methods, consideration of new approaches for adventitious agent testing and viral clearance studies, and establishing stability claim with less real-time data are additional components that enable an accelerated successful gene to clinical-grade material development strategy.

Antibody-drug conjugates come of age in oncology.

Antibody-drug conjugates (ADCs) combine the specificity of monoclonal antibodies with the potency of highly cytotoxic agents, potentially reducing the severity of side effects by preferentially targeting their payload to the tumour site. ADCs are being increasingly used in combination with other agents, including as first-line cancer therapies. As the technology to produce these complex therapeutics has matured, many more ADCs have been approved or are in late-phase clinical trials. The diversification of antigenic targets as well as bioactive payloads is rapidly broadening the scope of tumour indications for ADCs. Moreover, novel vector protein formats as well as warheads targeting the tumour microenvironment are expected to improve the intratumour distribution or activation of ADCs, and consequently their anticancer activity for difficult-to-treat tumour types. However, toxicity remains a key issue in the development of these agents, and better understanding and management of ADC-related toxicities will be essential for further optimization. This Review provides a broad overview of the recent advances and challenges in ADC development for cancer treatment.

10th antibody industrial symposium: new developments in antibody and adoptive cell therapies.

The annual "Antibody Industrial Symposium", co-organized by LabEx MAbImprove and MabDesign, held its 10th anniversary edition in Montpellier, France, on June 28-29, 2022. The meeting focused on new results and concepts in antibody engineering (naked, mono- or multi-specific, conjugated to drugs or radioelements) and also on new cell-based therapies, such as chimeric antigenic receptor (CAR)-T cells. The symposium, which brought together scientists from academia and industry, also addressed issues concerning the production of these molecules and cells, and the necessary steps to ensure a strong intellectual property protection of these new molecules and approaches. These two days of exchanges allowed a rich discussion among the various actors in the field of therapeutic antibodies.

Advanced mass spectrometry workflows for accurate quantification of trace-level host cell proteins in drug products: Benefits of FAIMS separation and gas-phase fractionation DIA.

Therapeutic monoclonal antibodies (mAb) production relies on multiple purification steps before release as a drug product (DP). A few host cell proteins (HCPs) may co-purify with the mAb. Their monitoring is crucial due to the considerable risk they represent for mAb stability, integrity, and efficacy and their potential immunogenicity. Enzyme-linked immunosorbent assays (ELISA) commonly used for global HCP monitoring present limitations in terms of identification and quantification of individual HCPs. Therefore, liquid chromatography tandem mass spectrometry (LC-MS/MS) has emerged as a promising alternative. Challenging DP samples show an extreme dynamic range requiring high performing methods to detect and reliably quantify trace-level HCPs. Here, we investigated the benefits of adding high-field asymmetric ion mobility spectrometry (FAIMS) separation and gas phase fractionation (GPF) prior to data independent acquisition (DIA). FAIMS LC-MS/MS analysis allowed the identification of 221 HCPs among which 158 were reliably quantified for a global amount of 880 ng/mg of NIST mAb Reference Material. Our methods have also been successfully applied to two FDA/EMA approved DPs and allowed digging deeper into the HCP landscape with the identification and quantification of a few tens of HCPs with sensitivity down to the sub-ng/mg of mAb level.

Interlaboratory Evaluation of a User-Friendly Benchtop Mass Spectrometer for Multiple-Attribute Monitoring Studies of a Monoclonal Antibody.

In the quest to market increasingly safer and more potent biotherapeutic proteins, the concept of the multi-attribute method (MAM) has emerged from biopharmaceutical companies to boost the quality-by-design process development. MAM strategies rely on state-of-the-art analytical workflows based on liquid chromatography coupled to mass spectrometry (LC-MS) to identify and quantify a selected series of critical quality attributes (CQA) in a single assay. Here, we aimed at evaluating the repeatability and robustness of a benchtop LC-MS platform along with bioinformatics data treatment pipelines for peptide mapping-based MAM studies using standardized LC-MS methods, with the objective to benchmark MAM methods across laboratories, taking nivolumab as a case study. Our results evidence strong interlaboratory consistency across LC-MS platforms for all CQAs (i.e., deamidation, oxidation, lysine clipping and glycosylation). In addition, our work uniquely highlights the crucial role of bioinformatics postprocessing in MAM studies, especially for low-abundant species quantification. Altogether, we believe that MAM has fostered the development of routine, robust, easy-to-use LC-MS platforms for high-throughput determination of major CQAs in a regulated environment.

Benefits and Limitations of High-Resolution Cyclic IM-MS for Conformational Characterization of Native Therapeutic Monoclonal Antibodies.

Monoclonal antibodies (mAbs) currently represent the main class of therapeutic proteins. mAbs approved by regulatory agencies are selected from IgG1, IgG2, and IgG4 subclasses, which possess different interchain disulfide connectivities. Ion mobility coupled to native mass spectrometry (IM-MS) has emerged as a valuable approach to tackle the challenging characterization of mAbs' higher order structures. However, due to the limited resolution of first-generation IM-MS instruments, subtle conformational differences on large proteins have long been hard to capture. Recent technological developments have aimed at increasing available IM resolving powers and acquisition mode capabilities, namely, through the release of high-resolution IM-MS (HR-IM-MS) instruments, like cyclic IM-MS (cIM-MS). Here, we outline the advantages and drawbacks of cIM-MS for better conformational characterization of intact mAbs (∼150 kDa) in native conditions compared to first-generation instruments. We first assessed the extent to which multipass cIM-MS experiments could improve the separation of mAbs' conformers. These initial results evidenced some limitations of HR-IM-MS for large native biomolecules which possess rich conformational landscapes that remain challenging to decipher even with higher IM resolving powers. Conversely, for collision-induced unfolding (CIU) approaches, higher resolution proved to be particularly useful (i) to reveal new unfolding states and (ii) to enhance the separation of coexisting activated states, thus allowing one to apprehend gas-phase CIU behaviors of mAbs directly at the intact level. Altogether, this study offers a first panoramic overview of the capabilities of cIM-MS for therapeutic mAbs, paving the way for more widespread HR-IM-MS/CIU characterization of mAb-derived formats.

Risk-Based Control Strategies of Recombinant Monoclonal Antibody Charge Variants.

Since the first approval of the anti-CD3 recombinant monoclonal antibody (mAb), muromonab-CD3, a mouse antibody for the prevention of transplant rejection, by the US Food and Drug Administration (FDA) in 1986, mAb therapeutics have become increasingly important to medical care. A wealth of information about mAbs regarding their structure, stability, post-translation modifications, and the relationship between modification and function has been reported. Yet, substantial resources are still required throughout development and commercialization to have appropriate control strategies to maintain consistent product quality, safety, and efficacy. A typical feature of mAbs is charge heterogeneity, which stems from a variety of modifications, including modifications that are common to many mAbs or unique to a specific molecule or process. Charge heterogeneity is highly sensitive to process changes and thus a good indicator of a robust process. It is a high-risk quality attribute that could potentially fail the specification and comparability required for batch disposition. Failure to meet product specifications or comparability can substantially affect clinical development timelines. To mitigate these risks, the general rule is to maintain a comparable charge profile when process changes are inevitably introduced during development and even after commercialization. Otherwise, new peaks or varied levels of acidic and basic species must be justified based on scientific knowledge and clinical experience for a specific molecule. Here, we summarize the current understanding of mAb charge variants and outline risk-based control strategies to support process development and ultimately commercialization.

Structural Analysis of Monoclonal Antibodies with Top-down and Middle-down Electron Transfer Dissociation Mass Spectrometry: The First Decade.

Monoclonal antibodies (mAbs) are protein biotherapeutics with a proven efficacy toward fighting life-threatening diseases. Their exceptional healing potential drives the annual increase in the number of novel mAbs and other antibody-like molecules entering clinical trials and the number of approved mAb-based drugs. Mass spectrometry (MS) offers high selectivity and specificity for the potentially unambiguous identification and comprehensive structural characterization of proteins, including at the proteoform level. It is thus not surprising that MS-based approaches are playing a central role in the biopharma laboratories, complementing and advancing traditional biotherapeutics characterization workflows. A combination of MS approaches is required to comprehensively characterize mAbs' structures: the commonly employed bottom-up MS approaches are efficiently complemented with mass measurements at the intact and subunit (middle-up) levels, together with product ion analysis following gas-phase fragmentation of precursor ions performed at the intact (top-down) and subunit (middle-down) levels. Here we overview our group's contribution to increasing the efficiency of these approaches and the development of the novel strategies over the past decade. Our particular focus has been on the top-down and middle-down MS methods that utilize electron transfer dissociation (ETD) for gas-phase protein ion fragmentation. Several approaches pioneered by our group, particularly an ETD-based middle-down approach, constitute a part of commercial software solutions for the mAb's characterization workflows.

Use of PASEF for Accelerated Protein Sequence Confirmation and De Novo Sequencing with High Data Quality.

Biopharmaceutical sequences can be well confirmed by multiple protease digests-e.g., trypsin, elastase, and chymotrypsin-followed by LC-MS/MS data analysis. High quality data can be used for de novo sequencing as well. PASEF (Parallel Accumulation and Serial Fragmentation) on the timsTOF instrument has been used to accelerate proteome and protein sequence studies and increase sequence coverage concomitantly.Here we describe the protein chemical and LC-MS methods in detail to generate high quality samples for sequence characterization from only 3 digests. We applied PASEF to generate exhaustive protein sequence coverage maps by combination of results from the three enzyme digests using a short LC gradient. The data quality obtained was high and adequate for determining antibody sequences de novo.Nivolumab and dulaglutide were digested by 3 enzymes individually. For nivolumab, 94/94/90% sequence coverage and 86/84/85% fragment coverage were obtained from the individual digest analysis with trypsin/chymotrypsin/elastase, respectively. For dulaglutide, 96/100/90% sequence coverage and 92/90/83% fragment coverage were obtained. The merged peptide map from the 3 digests for nivolumab resulted in ∼550 peptides; enough to safely confirm the full sequences and to determine the nivolumab sequence de novo.

Bispecific antibody characterization by a combination of intact and site-specific/chain-specific LC/MS techniques.

Bispecific antibodies (bsAbs) are considered as an important class of biopharmaceutical drugs, with about 160 products in clinical trials. From an analytical point of view, the correct chain-association is one of the most critical challenge to monitor during bsAbs development and production. In the present study, a full analytical workflow has been developed based on the use of various chromatographic modes: size exclusion chromatography (SEC), ion exchange chromatography (IEX), reversed phase liquid chromatography (RPLC), and hydrophilic interaction chromatography (HILIC), all combined with high resolution mass spectrometry (MS). This analytical strategy was applied to Hemlibra® (emicizumab), which is certainly the most successful commercial bsAb to date. Using this strategy, it was possible to monitor the presence of mispaired bsAb species and detect and identify additional post-translational modifications (PTMs).

Quantitative N-Glycan Profiling of Therapeutic Monoclonal Antibodies Performed by Middle-Up Level HILIC-HRMS Analysis.

The identification and accurate quantitation of the various glycoforms contained in therapeutic monoclonal antibodies (mAbs) is one of the main analytical needs in the biopharmaceutical industry, and glycosylation represents a crucial critical quality attribute (CQA) that needs to be addressed. Currently, the reference method for performing such identification/quantitation consists of the release of the N-glycan moieties from the mAb, their labelling with a specific dye (e.g., 2-AB or RFMS) and their analysis by HILIC-FLD or HILIC-MS. In this contribution, the potential of a new cost- and time-effective analytical approach performed at the protein subunit level (middle-up) was investigated for quantitative purposes and compared with the reference methods. The robustness of the approach was first demonstrated by performing the relative quantification of the glycoforms related to a well characterized mAb, namely adalimumab. Then, the workflow was applied to various glyco-engineered mAb products (i.e., obinutuzumab, benralizumab and atezolizumab). Finally, the glycosylation pattern of infliximab (Remicade®) was assessed and compared to two of its commercially available biosimilars (Remsima® and Inflectra®). The middle-up analysis proved to provide accurate quantitation results and has the added potential to be used as multi-attribute monitoring method.

Ultra-short ion-exchange columns for fast charge variants analysis of therapeutic proteins.

The purpose of this work was to study the potential of recently developed ultra-short column hardware for ion exchange chromatography (IEX). Various prototype and commercial columns having lengths of 5, 10, 15, 20 and 50 mm and packed with non-porous 3 µm particles were systematically compared. Both pH and salt gradient modes of elution were evaluated. Similarly, what has been previously reported for reversed phase liquid chromatography (RPLC) mode, an "on-off" retention mechanism was observed in IEX for therapeutic proteins and their fragments (25-150 kDa range). Because of the non-porous nature of the IEX packing material, the column porosity was relatively low (ε = 0.42) and therefore the volumes of ultra-short columns were very small. Based on this observation, it was important to reduce as much as possible all the sources of extra-column volumes (i.e. injection volume, extra-bed volume, detector cell volume and connector tubing volume), to limit peak broadening. With a fully optimized UHPLC system, very fast separations of intact and IdeS digested mAb products were successfully performed in about 1 min using an IEX column with dimensions of 15 × 2.1 mm. This column was selected for high-throughput separations, since it probably offers the best compromise between efficiency and analysis time. For such ultra-fast separations, PEEK tubing was applied to bypass the column oven (column directly connected) to the optical detector via a zero dead volume connection.

Towards a simple on-line coupling of ion exchange chromatography and native mass spectrometry for the detailed characterization of monoclonal antibodies.

This work describes the direct hyphenation of cation exchange chromatography (CEX) with a compact, easy-to-use benchtop Time of Flight mass spectrometer (ToF/MS) for the analytical characterization of monoclonal antibodies (mAbs). For this purpose, a wide range of commercial mAb products (including expired samples and mAb biosimilars) were selected to draw reliable conclusions. From a chromatographic point of view, various buffers and column dimensions were tested. When considering pH response, buffer stability over time and MS compatibility, the best compromise is represented by the following recipe: 50 mM ammonium acetate, titrated to pH 5.0 (mobile phase A) and 160 mM ammonium acetate, titrated to pH 8.5 (mobile phase B). Despite the broader peaks observed with the 2.1 mm i.d. CEX column, this was preferentially selected for CEX-MS operation, since the efficiency loss (caused by extra-column dispersion) was still acceptable while MS compatibility was strongly enhanced (thanks to low flow rate). In terms of MS, it was important to avoid the use of glass-bottled mobile phases, laboratory glassware and glass vials to minimize loss of MS resolution, sensitivity, and mass accuracy due to metal contaminants. With this new CEX-MS setup, straightforward and rapid analysis (in less than 10 min) of charge variants was possible, allowing the separation and identification of several charge variants.

High-Resolution IMS-MS to Assign Additional Disulfide Bridge Pairing in Complementarity-Determining Regions of an IgG4 Monoclonal Antibody.

Monoclonal antibodies (mAbs) have taken on an increasing importance for the treatment of various diseases, including cancers and immunological disorders. Disulfide bonds play a pivotal role in therapeutic antibody structure and activity relationships. Disulfide connectivity and cysteine-related variants are considered as critical quality attributes that must be monitored during mAb manufacturing and storage, as non-native disulfide bridges and aggregates might be responsible for loss of biological function and immunogenicity. The presence of cysteine residues in the complementarity-determining regions (CDRs) is rare in human antibodies but may be critical for the antigen-binding or deleterious for therapeutic antibody development. Consequently, in-depth characterization of their disulfide network is a prerequisite for mAb developability assessment. Mass spectrometry (MS) techniques represent powerful tools for accurate identification of disulfide connectivity. We report here on the MS-based characterization of an IgG4 comprising two additional cysteine residues in the CDR of its light chain. Classical bottom-up approaches after trypsin digestion first allowed identification of a dipeptide containing two disulfide bridges. To further investigate the conformational heterogeneity of the disulfide-bridged dipeptide, we performed ion mobility spectrometry-mass spectrometry (IMS-MS) experiments. Our results highlight benefits of high resolution IMS-MS to tackle the conformational landscape of disulfide peptides generated after trypsin digestion of a humanized IgG4 mAb under development. By comparing arrival time distributions of the mAb-collected and synthetic peptides, cyclic IMS afforded unambiguous assessment of disulfide bonds. In addition to classical peptide mapping, qualitative high-resolution IMS-MS can be of great interest to identify disulfide bonds within therapeutic mAbs.

State-of-the-Art Native Mass Spectrometry and Ion Mobility Methods to Monitor Homogeneous Site-Specific Antibody-Drug Conjugates Synthesis.

Antibody-drug conjugates (ADCs) are biotherapeutics consisting of a tumor-targeting monoclonal antibody (mAb) linked covalently to a cytotoxic drug. Early generation ADCs were predominantly obtained through non-selective conjugation methods based on lysine and cysteine residues, resulting in heterogeneous populations with varying drug-to-antibody ratios (DAR). Site-specific conjugation is one of the current challenges in ADC development, allowing for controlled conjugation and production of homogeneous ADCs. We report here the characterization of a site-specific DAR2 ADC generated with the GlyCLICK three-step process, which involves glycan-based enzymatic remodeling and click chemistry, using state-of-the-art native mass spectrometry (nMS) methods. The conjugation process was monitored with size exclusion chromatography coupled to nMS (SEC-nMS), which offered a straightforward identification and quantification of all reaction products, providing a direct snapshot of the ADC homogeneity. Benefits of SEC-nMS were further demonstrated for forced degradation studies, for which fragments generated upon thermal stress were clearly identified, with no deconjugation of the drug linker observed for the T-GlyGLICK-DM1 ADC. Lastly, innovative ion mobility-based collision-induced unfolding (CIU) approaches were used to assess the gas-phase behavior of compounds along the conjugation process, highlighting an increased resistance of the mAb against gas-phase unfolding upon drug conjugation. Altogether, these state-of-the-art nMS methods represent innovative approaches to investigate drug loading and distribution of last generation ADCs, their evolution during the bioconjugation process and their impact on gas-phase stabilities. We envision nMS and CIU methods to improve the conformational characterization of next generation-empowered mAb-derived products such as engineered nanobodies, bispecific ADCs or immunocytokines.

Fast Afucosylation Profiling of Glycoengineered Antibody Subunits by Middle-Up Mass Spectrometry.

Middle-up LC-MS antibody characterization workflows using reduction or IdeS digestion for a focused assessment of N-glycan profiling of three representative glycoengineered monoclonal antibodies (mAbs), namely, obinutuzumab (GlycomAb technology, Glycart/Roche), benralizumab (Potelligent Technology, BioWa, Kyowa Kirin) and mAb B (kifunensine) and compared to mAb A, produced in a common CHO cell line. In addition, EndoS or EndoS2 enzyme are used for quantitative determination of Fc-glycan core afucosylation and high mannose for these antibodies, as requested by health authorities for Fc-competent therapeutics mAbs critical quality attributes (CQAs).

Analysis of Monoclonal Antibody Glycopeptides by Capillary Electrophoresis-Mass Spectrometry Coupling (CE-MS).

Glycosylation is a crucial posttranslational modification (PTM) that might affect the safety and efficacy of monoclonal antibodies (mAbs). Capillary electrophoresis-mass spectrometry (CE-MS) enables the characterization of the primary structure of mAbs. A bottom-up proteomic workflow is designed to provide detailed information about the glycosylation. In this chapter, we describe the validated experimental protocol applied for the characterization and relative quantification of mAbs N-glycosylation at the glycopeptide level.

Alternative mobile phase additives for the characterization of protein biopharmaceuticals in liquid chromatography - Mass spectrometry.

When analyzing large complex protein biopharmaceuticals, ion-pairing agents imparting low pH are widely used as mobile phase additives to improve the chromatographic performance. However, one of the most effective additives in RPLC and HILIC, trifluoroacetic acid (TFA), is known as a strong suppressor of the MS signal and limits its use in hyphenated techniques. In this study, we evaluated a wide range of acidic additives to find alternatives to TFA that provided comparable chromatographic performance and improved MS sensitivity. It was observed that stronger acidic additives were required for intact level analysis compared to subunit level analysis and that the additive nature had a larger impact on the chromatographic performance in HILIC mode compared to RPLC. Therefore, four additives were identified as valuable alternatives to TFA in RPLC mode, namely, difluoroacetic acid (DFA), dichloroacetic acid (DClAA), trichloroacetic acid (TClAA), and methanesulfonic acid (MSA). Only one of these additives provided acceptable performance in HILIC mode, namely, TClAA. After evaluation of the MS performance, TClAA was discarded due to the apparent loss of intensity in both RPLC-MS and HILIC-MS mode. Together, these results demonstrate that for HILIC-MS analysis TFA remains the gold standard additive. However, DFA was found as promising alternative to TFA for RPLC-MS analysis and could play an important role in the development of methods for the characterization of the increasingly complex protein biopharmaceuticals.

New wide-pore superficially porous stationary phases with low hydrophobicity applied for the analysis of monoclonal antibodies.

The article describes the development of new stationary phases for the analysis of proteins in reversed phase liquid chromatography (RPLC). The goal was to have columns offering high recovery at low temperature, low hydrophobicity and novel selectivity. For this purpose, three different ligands bound onto the surface of superficially porous silica-based particles were compared, including trimethyl-silane (C1), ethyl-dimethyl-silane (C2) and N-(trifluoroacetomidyl)-propyl-diisopropylsilane (ES-LH). These three phases were compared with two commercial RPLC phases. In terms of protein recovery, the new ES-LH stationary phase clearly outperforms the other phases for any type of biopharmaceutical sample, and can already be successfully used at a temperature of only 60°C. In terms of retention, the new ES-LH and C1 materials were the less retentive ones, requiring lower organic solvent in the mobile phase. However, it is important to mention that the stability of C1 phase was critical under acidic, high temperature conditions. Finally, some differences were observed in terms of selectivity, particularly for the ES-LH column. Besides the chemical nature of the stationary phase, it was found that the nature of organic modifier also plays a key role in selectivity.