Rapid comparison of a candidate biosimilar to an innovator monoclonal antibody with advanced liquid chromatography and mass spectrometry technologies

This study shows that state-of-the-art liquid chromatography (LC) and mass spectrometry (MS) can be used for rapid verification of identity and characterization of sequence variants and posttranslational modifications (PTMs) for antibody products. A candidate biosimilar IgG1 monoclonal antibody (mAb) was compared in detail to a commercially available innovator product. Intact protein mass, primary sequence, PTMs, and the micro-differences between the two mAbs were identified and quantified simultaneously. Although very similar in terms of sequences and modifications, a mass difference observed by LC-MS intact mass measurements indicated that they were not identical. Peptide mapping, performed with data independent acquisition LC-MS using an alternating low and elevated collision energy scan mode (LC-MS^E), located the mass difference between the biosimilar and the innovator to a two amino acid residue variance in the heavy chain sequences. The peptide mapping technique was also used to comprehensively catalogue and compare the differences in PTMs of the biosimilar and innovator mAbs. Comprehensive glycosylation profiling confirmed that the proportion of individual glycans was different between the biosimilar and the innovator, although the number and identity of glycans were the same. These results demonstrate that the combination of accurate intact mass measurement, released glycan profiling, and LC-MS^E peptide mapping provides a set of routine tools that can be used to comprehensively compare a candidate biosimilar and an innovator mAb.     

Versatile characterization of glycosylation modification in CTLA4-Ig fusion proteins by liquid chromatography-mass spectrometry

CTLA4-Ig is a highly glycosylated therapeutic fusion protein that contains multiple N- and O-glycosylation sites. Glycosylation plays a vital role in protein solubility, stability, serum half-life, activity, and immunogenicity. For a CTLA4-Ig biosimilar development program, comparative analytical data, especially the glycosylation data, can influence decisions about the type and amount of animal and clinical data needed to establish biosimilarity. Because of the limited clinical experience with biosimilars before approval, a comprehensive level of knowledge about the biosimilar candidates is needed to achieve subsequent development. Liquid chromatography-mass spectrometry (LC–MS) is a versatile technique for characterizing N- and O-glycosylation modification of recombinant therapeutic proteins, including 3 levels: intact protein analysis, peptide mapping analysis, and released glycans analysis. In this report, an in-depth characterization of glycosylation of a candidate biosimilar was carried out using a systematic approach: N- and O-linked glycans were identified and electron-transfer dissociation was then used to pinpoint the 4 occupied O-glycosylation sites for the first time. As the results show, the approach provides a set of routine tools that combine accurate intact mass measurement, peptide mapping, and released glycan profiling. This approach can be used to comprehensively research a candidate biosimilar Fc-fusion protein and provides a basis for future studies addressing the similarity of CTLA4-Ig biosimilars.     

Versatile characterization of glycosylation modification in CTLA4-Ig fusion proteins by liquid chromatography-mass spectrometry

CTLA4-Ig is a highly glycosylated therapeutic fusion protein that contains multiple N- and O-glycosylation sites. Glycosylation plays a vital role in protein solubility, stability, serum half-life, activity, and immunogenicity. For a CTLA4-Ig biosimilar development program, comparative analytical data, especially the glycosylation data, can influence decisions about the type and amount of animal and clinical data needed to establish biosimilarity. Because of the limited clinical experience with biosimilars before approval, a comprehensive level of knowledge about the biosimilar candidates is needed to achieve subsequent development. Liquid chromatography-mass spectrometry (LC–MS) is a versatile technique for characterizing N- and O-glycosylation modification of recombinant therapeutic proteins, including 3 levels: intact protein analysis, peptide mapping analysis, and released glycans analysis. In this report, an in-depth characterization of glycosylation of a candidate biosimilar was carried out using a systematic approach: N- and O-linked glycans were identified and electron-transfer dissociation was then used to pinpoint the 4 occupied O-glycosylation sites for the first time. As the results show, the approach provides a set of routine tools that combine accurate intact mass measurement, peptide mapping, and released glycan profiling. This approach can be used to comprehensively research a candidate biosimilar Fc-fusion protein and provides a basis for future studies addressing the similarity of CTLA4-Ig biosimilars.     

Resolving the micro-heterogeneity and structural integrity of monoclonal antibodies by hybrid mass spectrometric approaches

For therapeutic monoclonal antibodies (mAbs), detailed analysis of the structural integrity and heterogeneity, which results from multiple types of post-translational modifications (PTMs), is relevant to various processes, including product characterization, storage stability and quality control. Despite the recent rapid development of new bioanalytical techniques, it is still challenging to completely characterize the proteoform profile of a mAb. As a nearly indispensable tool in mAb analysis, mass spectrometry (MS) provides unique structural information at multiple levels. Here, we tested a hybrid strategy for the comprehensive characterization of micro-heterogeneity by integrating 2 state-of-the-art MS-based approaches, high-resolution native MS and targeted glycan profiling, to perform complementary analysis at the intact protein level and released glycan level, respectively. We compared the performance of these methods using samples of engineered half-body IgG4s and a panel of mAbs approved for human use. The glycosylation characterization data derived from these approaches were found to be mutually consistent in composition profiling, and complementary in identification and relative-quantitation of low-abundant uncommon glycoforms. In addition, multiple other sources of micro-heterogeneity, such as glycation, lack of glycosylation, and loss of light chains, could be detected by this approach, and the contribution of multiple types of modifications to the overall micro-heterogeneity could be assessed using our superposition algorithm. Our data demonstrate that the hybrid strategy allows reliable and thorough characterization of mAbs, revealing product characteristics that would easily be missed if only a single approach were used.     

Advanced assessment of the physicochemical characteristics of Remicade® and Inflectra® by sensitive LC/MS techniques

In this study, we demonstrate the utility of ultra-performance liquid chromatography coupled to mass spectrometry (MS) and ion-mobility spectrometry (IMS) to characterize and compare reference and biosimilar monoclonal antibodies (mAbs) at an advanced level. Specifically, we focus on infliximab and compared the glycan profiles, higher order structures, and their host cell proteins (HCPs) of the reference and biosimilar products, which have the brand names Remicade® and Inflectra®, respectively. Overall, the biosimilar attributes mirrored those of the reference product to a very high degree. The glycan profiling analysis demonstrated a high degree of similarity, especially among the higher abundance glycans. Some differences were observed for the lower abundance glycans. Glycans terminated with N-glycolylneuraminic acid were generally observed to be at higher normalized abundance levels on the biosimilar mAb, while those possessing α-linked galactose pairs were more often expressed at higher levels on the reference molecule. Hydrogen deuterium exchange (HDX) analyses further confirmed the higher-order similarity of the 2 molecules. These results demonstrated only very slight differences between the 2 products, which, interestingly, seemed to be in the area where the N-linked glycans reside. The HCP analysis by a 2D-UPLC IMS-MS approach revealed that the same 2 HCPs were present in both mAb samples. Our ability to perform these types of analyses and acquire insightful data for biosimilarity assessment is based upon our highly sensitive UPLC MS and IMS methods.     

Controlled glycosylation of therapeutic antibodies in plants

Recombinant therapeutic monoclonal antibodies (mAb) can be expressed, assembled, and glycosylated in plants. Transgenic plants, producing anti-rabies mAb and anti-colorectal cancer mAb, were obtained from Agrobacterium-mediated transformation. The heavy chain (HC) of anti-rabies mAb was fused to the Lys-Asp-Glu-Leu (KDEL) endoplasmic reticulum retention signal whereas the HC of anti-colorectal cancer mAb was not fused to the KDEL sequence. Gel release of glycans and detection by high-performance liquid chromatography (HPLC), together with computer assisted analysis and matrix-assisted laser desorption/ionization time-of-flight (MALD-TOF) mass spectrometry, revealed that the plant-derived anti-rabies mAb with KDEL contained mainly oligomannose type N-glycans while the plant-derived anti-colorectal cancer mAb carried mainly biantennary glycans with and without a pentose sugar, that is thought to be xylose. This finding indicates that the KDEL sequence can affect the N-glycosylation processing of antibody in plant cells. The plant-derived mAbs with addition of a KDEL sequence did not contain any of the known antigenic glycan epitopes that are frequently found in other plant glycans or in mammalian-derived mAbs. The altered glycosylation on both plant-derived mAbs did not affect the activities that are required for therapy. These results indicate that plant genetic engineering could provide an effective and inexpensive means to control the glycosylation of therapeutic proteins such as mAbs, by the addition of a KDEL signal as a regulatory element.     

Characterization and comparison of commercially available TNF receptor 2-Fc fusion protein products

Because of rapidly increasing market demand and rising cost pressure, the innovator of etanercept (Enbrel®) will inevitably face competition from biosimilar versions of the product. In this study, to elucidate the differences between the reference etanercept and its biosimilars, we characterized and compared the quality attributes of two commercially available, biosimilar TNF receptor 2-Fc fusion protein products. Biosimilar 1 showed high similarity to Enbrel® in critical quality attributes including peptide mapping, intact mass, charge variant, purity, glycosylation and bioactivity. In contrast, the intact mass and MS/MS analysis of biosimilar 2 revealed a mass difference indicative of a two amino acid residue variance in the heavy chain (Fc) sequences. Comprehensive glycosylation profiling confirmed that biosimilar 2 has significantly low sialylated N-oligosaccharides. Biosimilar 2 also displayed significant differences in charge attributes compared with the reference product. Interestingly, biosimilar 2 exhibited similar affinity and bioactivity levels compared with the reference product despite the obvious difference in primary structure and partial physiochemical properties. For a biosimilar development program, comparative analytical data can influence decisions about the type and amount of animal and clinical data needed to demonstrate biosimilarity. Because of the limited clinical experience with biosimilars at the time of their approval, a thorough knowledge surrounding biosimilars and a case-by-case approach are needed to ensure the appropriate use of these products.     

Characterization and comparison of commercially available TNF receptor 2-Fc fusion protein products

Because of rapidly increasing market demand and rising cost pressure, the innovator of etanercept (Enbrel®) will inevitably face competition from biosimilar versions of the product. In this study, to elucidate the differences between the reference etanercept and its biosimilars, we characterized and compared the quality attributes of two commercially available, biosimilar TNF receptor 2-Fc fusion protein products. Biosimilar 1 showed high similarity to Enbrel® in critical quality attributes including peptide mapping, intact mass, charge variant, purity, glycosylation and bioactivity. In contrast, the intact mass and MS/MS analysis of biosimilar 2 revealed a mass difference indicative of a two amino acid residue variance in the heavy chain (Fc) sequences. Comprehensive glycosylation profiling confirmed that biosimilar 2 has significantly low sialylated N-oligosaccharides. Biosimilar 2 also displayed significant differences in charge attributes compared with the reference product. Interestingly, biosimilar 2 exhibited similar affinity and bioactivity levels compared with the reference product despite the obvious difference in primary structure and partial physiochemical properties. For a biosimilar development program, comparative analytical data can influence decisions about the type and amount of animal and clinical data needed to demonstrate biosimilarity. Because of the limited clinical experience with biosimilars at the time of their approval, a thorough knowledge surrounding biosimilars and a case-by-case approach are needed to ensure the appropriate use of these products.     

Correct primary structure assessment and extensive glyco-profiling of cetuximab by a combination of intact,middle-up,middle-down and bottom-up ESI and MALDI mass spectrometry techniques

The European Medicines Agency received recently the first marketing authorization application for a biosimilar monoclonal antibody (mAb) and adopted the final guidelines on biosimilar mAbs and Fc-fusion proteins. The agency requires high similarity between biosimilar and reference products for approval. Specifically, the amino acid sequences must be identical. The glycosylation pattern of the antibody is also often considered to be a very important quality attribute due to its strong effect on quality, safety, immunogenicity, pharmacokinetics and potency. Here, we describe a case study of cetuximab, which has been marketed since 2004. Biosimilar versions of the product are now in the pipelines of numerous therapeutic antibody biosimilar developers. We applied a combination of intact, middle-down, middle-up and bottom-up electrospray ionization and matrix assisted laser desorption ionization mass spectrometry techniques to characterize the amino acid sequence and major post-translational modifications of the marketed cetuximab product, with special emphasis on glycosylation. Our results revealed a sequence error in the reported sequence of the light chain in databases and in publications, thus highlighting the potency of mass spectrometry to establish correct antibody sequences. We were also able to achieve a comprehensive identification of cetuximab’s glycoforms and glycosylation profile assessment on both Fab and Fc domains. Taken together, the reported approaches and data form a solid framework for the comparability of antibodies and their biosimilar candidates that could be further applied to routine structural assessments of these and other antibody-based products.     

Monoclonal antibodies biosimilarity assessment using transient isotachophoresis capillary zone electrophoresis-tandem mass spectrometry

Out of all categories, monoclonal antibody (mAb) therapeutics attract the most interest due to their strong therapeutic potency and specificity. Six of the 10 top-selling drugs are antibody-based therapeutics that will lose patent protection soon. The European Medicines Agency has pioneered the regulatory framework for approval of biosimilar products and approved the first biosimilar antibodies by the end of 2013. As highly complex glycoproteins with a wide range of micro-variants, mAbs require extensive characterization through multiple analytical methods for structure assessment rendering manufacturing control and biosimilarity studies particularly product and time-consuming. Here, capillary zone electrophoresis coupled to mass spectrometry by a sheathless interface (CESI-MS) was used to characterize marketed reference mAbs and their respective biosimilar candidate simultaneously over different facets of their primary structure. CESI-MS/MS data were compared between approved mAbs and their biosimilar candidates to prove/disconfirm biosimilarity regarding recent regulation directives. Using only a single sample injection of 200 fmol, CESI-MS/MS data enabled 100% amino acids (AA) sequence characterization, which allows a difference of even one AA between 2 samples to be distinguished precisely. Simultaneously glycoforms were characterized regarding their structures and position through fragmentation spectra and glycoforms semiquantitative analysis was established, showing the capacity of the developed methodology to detect up to 16 different glycans. Other posttranslational modifications hotspots were characterized while their relative occurrence levels were estimated and compared to biosimilars. These results proved the value of using CESI-MS because the separation selectivity and ionization efficiency provided by the system allowed substantial improvement in the characterization workflow robustness and accuracy. Biosimilarity assessment could be performed routinely with a single injection of each candidate enabling improvements in the biosimilar development pipeline.     

Monoclonal antibodies biosimilarity assessment using transient isotachophoresis capillary zone electrophoresis-tandem mass spectrometry

Out of all categories, monoclonal antibody (mAb) therapeutics attract the most interest due to their strong therapeutic potency and specificity. Six of the 10 top-selling drugs are antibody-based therapeutics that will lose patent protection soon. The European Medicines Agency has pioneered the regulatory framework for approval of biosimilar products and approved the first biosimilar antibodies by the end of 2013. As highly complex glycoproteins with a wide range of micro-variants, mAbs require extensive characterization through multiple analytical methods for structure assessment rendering manufacturing control and biosimilarity studies particularly product and time-consuming. Here, capillary zone electrophoresis coupled to mass spectrometry by a sheathless interface (CESI-MS) was used to characterize marketed reference mAbs and their respective biosimilar candidate simultaneously over different facets of their primary structure. CESI-MS/MS data were compared between approved mAbs and their biosimilar candidates to prove/disconfirm biosimilarity regarding recent regulation directives. Using only a single sample injection of 200 fmol, CESI-MS/MS data enabled 100% amino acids (AA) sequence characterization, which allows a difference of even one AA between 2 samples to be distinguished precisely. Simultaneously glycoforms were characterized regarding their structures and position through fragmentation spectra and glycoforms semiquantitative analysis was established, showing the capacity of the developed methodology to detect up to 16 different glycans. Other posttranslational modifications hotspots were characterized while their relative occurrence levels were estimated and compared to biosimilars. These results proved the value of using CESI-MS because the separation selectivity and ionization efficiency provided by the system allowed substantial improvement in the characterization workflow robustness and accuracy. Biosimilarity assessment could be performed routinely with a single injection of each candidate enabling improvements in the biosimilar development pipeline.     

A review of methods for interpretation of glycopeptide tandem mass spectral data

Despite the publication of several software tools for analysis of glycopeptide tandem mass spectra, there remains a lack of consensus regarding the most effective and appropriate methods. In part, this reflects problems with applying standard methods for proteomics database searching and false discovery rate calculation. While the analysis of small post-translational modifications (PTMs) may be regarded as an extension of proteomics database searching, glycosylation requires specialized approaches. This is because glycans are large and heterogeneous by nature, causing glycopeptides to exist as multiple glycosylated variants. Thus, the mass of the peptide cannot be calculated directly from that of the intact glycopeptide. In addition, the chemical nature of the glycan strongly influences product ion patterns observed for glycopeptides. As a result, glycopeptidomics requires specialized bioinformatics methods. We summarize the recent progress towards a consensus for effective glycopeptide tandem mass spectrometric analysis.     

N-Glycan structure annotation of glycopeptides using a linearized glycan structure database (GlyDB)

While glycoproteins are abundant in nature, and changes in glycosylation occur in cancer and other diseases, glycoprotein characterization remains a challenge due to the structural complexity of the biopolymers. This paper presents a general strategy, termed GlyDB, for glycan structure annotation of N-linked glycopeptides from tandem mass spectra in the LC-MS analysis of proteolytic digests of glycoproteins. The GlyDB approach takes advantage of low-energy collision-induced dissociation of N-linked glycopeptides that preferentially cleaves the glycosidic bonds while the peptide backbone remains intact. A theoretical glycan structure database derived from biosynthetic rules for N-linked glycans was constructed employing a novel representation of branched glycan structures consisting of multiple linear sequences. The commonly used peptide identification program, Sequest, could then be utilized to assign experimental tandem mass spectra to individual glycoforms. Analysis of synthetic glycopeptides and well-characterized glycoproteins demonstrate that the GlyDB approach can be a useful tool for annotation of glycan structures and for selection of a limited number of potential glycan structure candidates for targeted validation.     

Intact glycopeptide characterization using mass spectrometry

Glycosylation is one of the most prominent and extensively studied protein post-translational modifications. However, traditional proteomic studies at the peptide level (bottom-up) rarely characterize intact glycopeptides (glycosylated peptides without removing glycans), so no glycoprotein heterogeneity information is retained. Intact glycopeptide characterization, on the other hand, provides opportunities to simultaneously elucidate the glycan structure and the glycosylation site needed to reveal the actual biological function of protein glycosylation. Recently, significant improvements have been made in the characterization of intact glycopeptides, ranging from enrichment and separation, mass spectroscopy (MS) detection, to bioinformatics analysis. In this review, we recapitulated currently available intact glycopeptide characterization methods with respect to their advantages and limitations as well as their potential applications.     

Mapping of conformational mAb epitopes to the C domain of human angiotensin I-converting enzyme

Angiotensin I-converting enzyme (ACE, CD143) has two homologous domains, each having a functional active site. Fine epitope mapping of 8 mAbs to the C-terminal domain of human ACE was carried out using plate precipitation assays, mAbs' cross-reactivity with ACE from different species, site-directed mutagenesis, and antigen- and cell-based ELISAs. Almost all epitopes contained potential glycosylation sites. Therefore, these mAbs could be used to distinguish different glycoforms of ACE expressed in different tissues or cell lines. mAbs 1B8 and 3F10 were especially sensitive to the composition of the N-glycan attached to Asn 731; mAbs 2H9 and 3F11 detected the glycosylation status of the glycan attached to Asn 685 and perhaps Asn1162; and mAb 1E10 and 4E3 recognized the glycan on Asn 666. The epitope of mAb 1E10 is located at the N-terminal end of the C domain, close to the unique 36 amino acid residues of testicular ACE (tACE). Moreover, it binds preferentially to tACE on the surface of human spermatozoa and thus may find application as an immunocontraceptive drug. mAb 4E3 was the best mAb for quantification of ACE-expressing somatic cells by flow cytometry. In contrast to the other mAbs, binding of mAb 2B11 was not markedly influenced by ACE glycosylation or by the cell culture conditions or cell types, making this mAb a suitable reference antibody. Epitope mapping of these C-domain mAbs, particularly those that compete with N-domain mAbs, enabled us to propose a model of the two-domain somatic ACE that might explain the interdomain cooperativity. Our findings demonstrated that mAbs directed to conformational epitopes on the C-terminal domain of human ACE are very useful for the detection of testicular and somatic ACE, quantification using flow cytometry and ELISA assays, and for the study of different aspects of ACE biology.     

Comprehensive glycan analysis of recombinant Aspergillus niger endo-polygalacturonase C

The enzyme PGC is produced by the fungus Aspergillus niger during invasion of plant cell walls. The enzyme has been homologously overexpressed to provide sufficient quantities of purified enzyme for biological studies. We have characterized this enzyme in terms of its posttranslational modifications (PTMs) and found it to be both N- and O-glycosylated. The glycosyl moieties have also been characterized. This has involved a combination of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF), liquid chromatography (LC)-ion trap, and LC-electrospray ionization (ESI) mass spectrometries in conjunction with trypsin degradation and beta-elimination, followed by Michael addition with dithiothreitol (BEMAD). This is the first demonstration of the ability of BEMAD to map glycosylation sites other than O-GlcNAc sites. The complete characterization of all PTMs on PGC allows us to model them on the peptide backbone, revealing potential roles played by the glycans in modulating the interaction of the enzyme with other macromolecules.     

Physicochemical Characterization,Glycosylation Pattern and Biosimilarity Assessment of the Fusion Protein Etanercept

Etanercept is a soluble fusion protein of the tumor necrosis factor receptor (TNFR) extracellular domain, linked to an Fc part of IgG1. It possesses three N- and 13 O-glycosylation sites. Due to its complex structure, an analytical challenge is facing the development and approval of biosimilars. In the current study, physicochemical characterization using state-of-the-art analytics was performed to analyze intact and subunit masses, post-translational modifications (PTMs), higher order structure and potency of Etanercept originator Enbrel^® and its biosimilar Altebrel? (AryoGen Pharmed) in accordance to critical quality attributes of biopharmaceuticals. Intact mass and subunit analysis revealed a size of about 126 kDa for both biologicals. Similar glycoprotein species for the complete monomer and the Fc domain of originator and follow-on product were observed, however, small differences in lysine variants and oxidation were found. N-Glycopeptide analysis with UHPLC-QTOF-MS^E confirmed the N-glycosylation sites (N149, N171 and N317) as well as Fc-specific glycosylation on N317, and TNFR-specific highly sialylated glycans on N149 and N171 on both investigated products. Small quantitative variations in the N-glycan profile were detected, although the N-glycans were qualitatively similar. Four different O-glycopeptides bearing core 1-type glycans were detected. For both, N- and O-glycopeptide analysis, determination was achieved without prior cleavage of the sialic acid residues for the first time. In addition, ion mobility spectrometry data confirmed close similarity of higher-order structure of both biologics. Furthermore, a neutralization assay, investigating the impact of altered PTMs on potency, indicated that the differences within all batches are still in the acceptable range for biosimilarity.     

综述: N-连接完整糖肽的质谱分析策略和研究方法

蛋白质糖基化作为最普遍、最重要的蛋白质修饰,一直是组学研究的焦点之一．近十几年来,N-连接糖蛋白质组学研究普遍采用的方法是将糖链与所修饰的多肽分开进行分析．该策略虽降低了分析难度,却也丢失了糖链与蛋白质糖基化位点间重要的对应关系信息．近年来,完整糖肽的质谱分析策略和方法逐步建立起来．总体而言,要实现对完整糖肽的直接质谱分析,首先需要从复杂样品中富集完整糖肽以消除非糖基化多肽对完整糖肽分析的影响,然后在质谱分析中还需要根据糖肽特性调整相应质谱分析参数,最后在后续数据分析中还需要开发相应的分析软件以完成完整糖肽中多肽序列和糖链组成或结构的鉴定．本文即从以上三个主要方面系统阐述目前N-完整糖肽分析中常用的质谱和数据分析策略和方法,并进一步在糖肽谱图识别、母离子单同位素分子质量校正、数据库选择以及假阳性率评估和控制等方面都进行了逐一探讨．完整糖肽的直接质谱分析有助于获取糖链和糖基化位点间的对应关系信息,可为生物标志物发现和疾病致病机理等研究提供更有力的糖蛋白质组学研究工具．     

Optimization of apolipoprotein-B-100 sequence coverage by liquid chromatography-tandem mass spectrometry for the future study of its posttranslational modifications

Proteomic applications have been increasingly used to study posttranslational modifications of proteins (PTMs). For the purpose of identifying and localizing specific but unknown PTMs on huge proteins, improving their sequence coverage is fundamental. Using liquid chromatography coupled to mass spectrometry (LC–MS/MS), peptide mapping of the native apolipoprotein-B-100 was performed to further document the effects of oxidation. Apolipoprotein-B-100 is the main protein of low-density lipoprotein particles and its oxidation could play a role in atherogenesis. Because it is one of the largest human proteins, the sequence recovery rate of apolipoprotein-B-100 only reached 1% when conventional analysis parameters were used. The different steps of the peptide mapping process—from protein treatment to data analysis—were therefore reappraised and optimized. These optimizations allowed a protein sequence recovery rate of 79%, a rate which has never been achieved previously for such a large human protein. The key points for improving peptide mapping were optimization of the data analysis software; peptide separation by LC; sample preparation; and MS acquisition. The new protocol has allowed us to increase by a factor of 4 the detection of modified peptides in apolipoprotein-B-100. This approach could easily be transferred to any study of PTMs using LC–MS/MS.     

The criticality of high-resolution N-linked carbohydrate assays and detailed characterization of antibody effector function in the context of biosimilar development

Accurate measurement and functional characterization of antibody Fc domain N-linked glycans is critical to successful biosimilar development. Here, we describe the application of methods to accurately quantify and characterize the N-linked glycans of 2 IgG1 biosimilars with effector function activity, and show the potential pitfalls of using assays with insufficient resolution. Accurate glycan assessment was combined with glycan enrichment using lectin chromatography or production with glycosylation inhibitors to produce enriched pools of key glycan species for subsequent assessment in cell-based antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity effector function assays. This work highlights the challenges of developing high-quality biosimilar candidates and the need for modern biotechnology capabilities. These results show that high-quality analytics, combined with sensitive cell-based assays to study in vivo mechanisms of action, is an essential part of biosimilar development.