Characterization Of Therapeutic Monoclonal Antibodies Research Articles

Advanced assessment through intact glycopeptide analysis of Infliximab's biologics and biosimilar.

Characterization of therapeutic monoclonal antibodies (mAbs) represents a major challenge for analytical sciences due to their heterogeneity associated with post-translational modifications (PTMs). The protein glycosylation requires comprehensive identification, which could influence on the mAbs' structure and their function. Here, we demonstrated high-resolution tandem mass spectrometry with an ultra-high-performance liquid chromatography for characterization and comparison between biologics and biosimilar of infliximab at an advanced level. Comparing the N- and O-glycopeptides profiles, a total of 49 and 54 glycopeptides was identified for each product of the biologics and biosimilar, respectively. We also discovered one novel N-glycosylation site at the light chain from both biopharmaceuticals and one novel O-glycopeptide at the heavy chain from only biosimilar. Site-specific glycopeptide analysis process will be a robust and useful technique for evaluating therapeutic mAbs and complex glycoprotein products.

Multiple-parallel-protease digestion coupled with high-resolution mass spectrometry: An approach towards comprehensive peptide mapping of therapeutic mAbs

Therapeutic monoclonal antibodies (mAbs) are structurally large and complex molecules. To be safe and efficacious, a biosimilar mAb must show high similarity to its reference product in Critical Quality Attributes (CQA). mAbs are highly sensitive to protein expression, production, manufacturing, supply chain, and storage conditions. All these factors make biosimilar mAbs intrinsically susceptible for variability during production. Accordingly, several lots of references and tests are required to establish the biosimilarity of a test mAb. The primary structure is a CQA of a mAb affecting its safety and efficacy. Here, we apply peptide mapping as an analytical method to decipher the primary structure and associated modifications for a quick quality assessment of TrastuzumAb and RituximAb innovator and biosimilar. A multiple-parallel-protease digestion strategy followed by high-resolution mass spectrometric analysis consistently achieved 100% sequence coverage along with reliable detection of post-translational modifications. Additionally, the use of supporting methods such as intact mass analysis and circular dichroism helped us to decipher the primary and higher order structures of these mAbs. We identify discernible variations in the profile of the innovator and biosimilar mAbs and validate the method for quick yet deep comparability analysis of the primary structure of biosimilar mAbs sold in the market. SignificancePeptide mapping using bottom-up approach is one of the most common methods for the characterization of therapeutic monoclonal antibodies. Herein, we describe a multi-parallel-protease digestion strategy using a combination of five different proteases followed by high-resolution mass spectrometric analysis with TrastuzumAb and RituximAb as an example. This resulted in a comprehensive identification of peptides with increased reliability and identification of different PTMs. Additional supporting orthogonal methods like intact mass and higher-order structure analysis helped evaluate broader conformational properties.

Towards middle-up analysis of polyclonal antibodies: subclass-specific N-glycosylation profiling of murine immunoglobulin G (IgG) by means of HPLC-MS

In recent years, advanced HPLC-MS strategies based on intact protein (“top-down”) or protein subunit (“middle-up/middle-down”) analysis have been implemented for the characterization of therapeutic monoclonal antibodies. Here, we assess feasibility of middle-up/middle-down analysis for polyclonal IgGs exhibiting extensive sequence variability. Specifically, we addressed IgGs from mouse, representing an important model system in immunological investigations. To obtain Fc/2 portions as conserved subunits of IgGs, we made use of the bacterial protease SpeB. For this purpose, we initially determined SpeB cleavage sites in murine IgGs. The resulting Fc/2 portions characteristic of different subclasses were subsequently analysed by ion-pair reversed-phase HPLC hyphenated to high-resolution mass spectrometry. This enabled simultaneous relative quantification of IgG subclasses and their N-glycosylation variants, both of which influence IgG effector functions. To assess method capabilities in an immunological context, we applied the analytical workflow to polyclonal antibodies obtained from BALB/c mice immunized with the grass pollen allergen Phl p 6. The study revealed a shift in IgG subclasses and Fc-glycosylation patterns in total and antigen-specific IgGs from different mouse cohorts, respectively. Eventually, Fc/2 characterization may reveal other protein modifications including oxidation, amino acid exchanges, and C-terminal lysine, and may thus be implemented for quality control of functional antibodies.

Protocols for the analytical characterization of therapeutic monoclonal antibodies. III – Denaturing chromatographic techniques hyphenated to mass spectrometry

The full analytical characterization of therapeutic monoclonal antibodies (mAbs) requires a large variety of complementary information that can be obtained by chromatographic methods. A series of protocols papers has been proposed to cover the chromatographic techniques and the enzymatic and chemical sample preparation procedures generally applied for the analytical characterization of therapeutic mAbs. The present protocol paper focuses on denaturing chromatographic techniques hyphenated to mass spectrometry, namely reversed-phase liquid chromatography (RPLC) and hydrophilic interaction chromatography (HILIC), to assess the subtle mAbs structural heterogeneity resulting from glycosylation patterns and post-transcriptional modifications (PTMs). In this paper, some generic protocols are provided, using a wide range of therapeutic mAbs approved by Food and Drug Administration (FDA) and European Medicines Agency (EMA), to illustrate the possibilities offered by the last generation of RPLC and HILIC columns when performing LC-MS analysis at the middle-up level.

Characterization of Therapeutic Monoclonal Antibodies by Using FcγR-expressing Reporter Cell Lines.

Fcγ receptors (FcγRs), which bind to the Fc regions of antibodies, play an important role in antibody effector functions. In humans, there are four types of activating FcγRs: FcγRI, FcγRIIa, FcγRIIIa, and FcγRIIIb. These are expressed on various effector cells such as natural killer (NK) cells, neutrophils and macrophages. FcγRIIIa expressed on NK cells is known to play a pivotal role in antibody-dependent cellular cytotoxicity (ADCC) by therapeutic monoclonal antibodies (mAbs). To assess the ADCC activity of mAbs, the killing of target cells is often measured using human peripheral mononuclear blood cells (hPBMCs) or isolated primary NK cells as effector cells. These assays can directly assess the cytotoxicity induced by mAbs, but require fresh blood from donors, and are insufficiently reproducible due to differences in effector cell activity among donors. We developed a cell-based assay using reporter cell lines expressing human FcγR and a nuclear factor of activated T cells (NFAT)-driven luciferase reporter gene (Jurkat/FcγR/NFAT-Luc), which can estimate the activation of various FcγRs by antigen-bound mAbs in vitro, with high reproducibility. The usefulness of this assay was confirmed by comparing mAbs activity with different abilities to activate FcγRs, including Fc-engineered anti-CD20 mAbs and anti-EGFR mAbs with different IgG subclasses. We also confirmed the application of this assay for the characterization of mAbs product-related substances. Our FcγR reporter assay is a promising new tool for the characterization of therapeutic mAbs in various stages of mAbs development.

Protocols for the analytical characterization of therapeutic monoclonal antibodies. II - Enzymatic and chemical sample preparation.

The analytical characterization of therapeutic monoclonal antibodies and related proteins usually incorporates various sample preparation methodologies. Indeed, quantitative and qualitative information can be enhanced by simplifying the sample, thanks to the removal of sources of heterogeneity (e.g. N-glycans) and/or by decreasing the molecular size of the tested protein by enzymatic or chemical fragmentation. These approaches make the sample more suitable for chromatographic and mass spectrometric analysis. Structural elucidation and quality control (QC) analysis of biopharmaceutics are usually performed at intact, subunit and peptide levels. In this paper, general sample preparation approaches used to attain peptide, subunit and glycan level analysis are overviewed. Protocols are described to perform tryptic proteolysis, IdeS and papain digestion, reduction as well as deglycosylation by PNGase F and EndoS2 enzymes. Both historical and modern sample preparation methods were compared and evaluated using rituximab and trastuzumab, two reference therapeutic mAb products approved by Food and Drug Administration (FDA) and European Medicines Agency (EMA). The described protocols may help analysts to develop sample preparation methods in the field of therapeutic protein analysis.

Protocols for the analytical characterization of therapeutic monoclonal antibodies. I - Non-denaturing chromatographic techniques.

Size-, charge- and hydrophobicity-related variants of a biopharmaceutical product have to be deeply characterized for batch consistency and for the assessment of immunogenicity and safety effects. Size exclusion chromatography (SEC) and ion exchange chromatography (IEX) are considered as the gold standard for the analysis of high molecular weight species (HMWS) and charge-related variants, respectively. Hydrophobic interaction chromatography (HIC) has drawn renewed attention to monitor the small drug payload distribution in the cysteine-linked antibody-drug conjugates (ADC). These three chromatographic techniques, namely SEC, HIC and IEX, are historical, non-denaturing and robust approaches widely used for the characterization of biopharmaceutical proteins. Despite the broad spectrum of monoclonal antibodies (mAbs) structures, isoelectric points (pIs) and hydrophobicities, generic protocols can be applied to separate their size-, charge- and hydrophobicity-related variants, using the last generation of chromatographic columns and appropriate mobile phase conditions. Straightforward protocols are described in this manuscript with representative chromatograms of ten distinct Food and Drug Administration (FDA) and European Medicines Agency (EMA) approved therapeutic mAb products to illustrate the performance of the SEC, IEX and HIC methods.

In vivo characterization of therapeutic monoclonal antibodies

Therapeutic monoclonal antibodies (mAbs) are complex drug molecules with a high degree of heterogeneity, including charge variants, aggregates, fragments, and post-translational modifications (PTMs). PTMs can be introduced by chemical or enzymatic modifications over the lifespan of mAbs during production, storage, and in vivo circulation. Common PTMs in mAbs include glycosylation, deamidation, oxidation, glycation, N-terminal glutamine cyclization, and C-terminal lysine processing. These product-related modifications are termed as product quality attributes (PQAs). A given PQA has the potential to impact the safety and/or efficacy of a therapeutic mAb. A PQA may enhance immunogenicity or off-target bindings, resulting in changes of the safety profile of a therapeutic mAb. A PQA may change the interaction of a therapeutic mAb to its target antigen, and therefore alter the efficacy. The PQAs that impact on drug safety and/or efficacy are considered as critical quality attributes (CQAs). Control of PQAs, especially CQAs, within predefined acceptance criteria is required by regulatory agencies to ensure product quality, consistency, and stability. During drug development, manufacturing processes aim to maximize the production of a desired product in high purity with minimal and consistent levels of variant forms. Formulation development seeks to stabilize mAb products and minimize additional formation of variants during storage. The modifications occurred during mAb production and storage can be reliably monitored and controlled. However, additional modifications can occur after a mAb is administered to patients. mAbs are typically administered intravenously or subcutaneously, and quickly get into the circulation of patients. The circulating half-life of a typical mAb in human blood system is 2-3 weeks. The blood environment is remarkably different from formulation buffers and storage temperatures. Typical mAb drug products are formulated at a pH of 5-6 with multiple excipients, such as sugars, salts, amino acids, and surfactants. The formulation buffer, pH, and excipients are optimized to enhance mAb solubility/stability and to reduce viscosity as well as the formation of aggregates/particulates. In contrast, blood is a dynamic environment with thousands of proteins including many protein-modifying enzymes, and is tightly regulated at a pH of 7.4 and a temperature of 37 °C. The clearance and modification of a mAb in blood circulation may result in changes of the PQA profile, leading to increased attribute exposure and decreased effective drug exposure to patients, and therefore causing the concerns of immune responses and loss of efficacy. To this end, a recent FDA guidance for industry recommends sponsors to evaluate susceptibilities of therapeutic protein products to modifications in the in vivo milieu as early as in product design and development, in order to facilitate product engineering for enhancing the product stability under in vivo conditions [1]. Therefore, in vitro and in vivo characterization of PQAs can monitor the attribute stability before and after drug administration and JOURNAL OF APPLIED BIOANALYSIS, January 2016, p. 10-15. http://dx.doi.org/10.17145/jab.16.003 (ISSN 2405-710X) Vol. 2, No. 1

High-throughput epitope binning of therapeutic monoclonal antibodies: why you need to bin the fridge

Analytical tools are evolving to meet the need for the higher-throughput characterization of therapeutic monoclonal antibodies. An antibody's epitope is arguably its most important property because it underpins its functional activity but, because epitope selection is innate, it remains an empirical process. Here, we focus on the emergence of label-free biosensors with throughput capabilities orders of magnitude higher than the previous state-of-the-art, which can facilitate large assays such as epitope binning so that they can be incorporated alongside functional activity screens, enabling the rapid identification of leads that exhibit unique and functional epitopes. In addition to streamlining the drug development process by saving time and cost, the information from epitope binning assays could provide the basis for intellectual property protection.

Development of a cell-based assay measuring the activation of FcγRIIa for the characterization of therapeutic monoclonal antibodies.

Antibody-dependent cellular cytotoxicity (ADCC) is one of the important mechanisms of action of the targeting of tumor cells by therapeutic monoclonal antibodies (mAbs). Among the human Fcγ receptors (FcγRs), FcγRIIIa is well known as the only receptor expressed in natural killer (NK) cells, and it plays a pivotal role in ADCC by IgG1-subclass mAbs. In addition, the contributions of FcγRIIa to mAb-mediated cytotoxicity have been reported. FcγRIIa is expressed in myeloid effector cells including neutrophils and macrophages, and it is involved in the activation of these effector cells. However, the measurement of the cytotoxicity via FcγRIIa-expressing effector cells is complicated and inconvenient for the characterization of therapeutic mAbs. Here we report the development of a cell-based assay using a human FcγRIIa-expressing reporter cell line. The FcγRIIa reporter cell assay was able to estimate the activation of FcγRIIa by antigen-bound mAbs by a very simple method in vitro. The usefulness of this assay for evaluating the activity of mAbs with different abilities to activate FcγRIIa was confirmed by the examples including the comparison of the activity of the anti-CD20 mAb rituximab and its Fc-engineered variants, and two anti-EGFR mAbs with different IgG subclasses, cetuximab (IgG1) and panitumumab (IgG2). We also applied this assay to the characterization of a force-oxidized mAb, and we observed that oxidation significantly decreased the FcγRIIa activation by EGFR-bound cetuximab. These results suggest that our FcγRIIa reporter assay is a promising tool for the characterization of therapeutic mAbs, including Fc-engineered mAbs, IgG2-subclass mAbs, and their product-related variants.

Characterization of Therapeutic Monoclonal Antibodies Reveals Differences Between In Vitro and In Vivo Time-Course Studies

To examine and determine the sites and the kinetics of IgG1 mAb modifications from both in vitro (rat plasma and PBS) and in vivo (rat model) time-course studies. A comprehensive set of protein characterization methods, including RPLC/MS, LC-MS/MS, iCIEF, capSEC, and CE-SDS were performed in this report. We demonstrate that plasma incubation and in vivo circulation increase the rate of C-terminal lysine removal, and the levels of deamidation, pyroglutamic acid (pyroE), and thioether-linked (lanthionine) heavy chain and light chain (HC-S-LC). In contrast, incubation in PBS shows no C-terminal lysine removal, and slower rates of deamidation, pyroE, and HC-S-LC formation. Other potential modifications such as oxidation, aggregation, and peptide bonds hydrolysis are not enhanced. This study demonstrates that in vivo mAb modifications are not fully represented by in vitro PBS or plasma incubation. The differences in modifications and their rates reflect the dissimilarities of matrices and the impact of enzymes. These observations provide valuable evidence and knowledge in evaluating the criticality of modifications that occur naturally in vivo that might impact formulation design, therapeutic outcome, and critical quality attribute assessments for therapeutic mAb manufacturing and quality control.

Characterization of Humanized Anti-Tac Monoclonal Antibody by Traditional Separation Techniques and Capillary Electrophoresis

Abstract Humanized anti-Tac monoclonal antibody has been characterized by both SDS-PAGE and isoelectric focusing using traditional polyacrylamide methods. In addition to these techniques, capillary electrophoresis has been adapted to isoelectric focusing in a liquid phase. The humanized antibody has a pl in the range of 8.2–9.0, a region in which precast polyacrylamide or agarose gels are not stable in the 7–10 pH range. Experiments have been conducted to generate gradients in the 3–10 pH range and these gradients have successfully separated differently charged subspecies of humanized anti-TAC. Because of the simplicity in generating a variety of gradients, the power of resolution in different modes, its high efficiency, and the minimum sample volume requirements, capillary electrophoresis may become the method of choice for the routine characterization of therapeutic monoclonal antibodies and their stability profiles.