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.     

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.Key words: biosimilar mAb, innovator mAb, molecular similarity, sequence variants, posttranslational modifications, N-linked glycosylation, chemical degradations, micro-heterogeneities, characterization, intact protein mass measurement, peptide mapping, glycan profiling, LC-MS, LC-fluorescence, MALDI MS     

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.     

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.     

Full validation of therapeutic antibody sequences by middle-up mass measurements and middle-down protein sequencing

The regulatory bodies request full sequence data assessment both for innovator and biosimilar monoclonal antibodies (mAbs). Full sequence coverage is typically used to verify the integrity of the analytical data obtained following the combination of multiple LC-MS/MS datasets from orthogonal protease digests (so called “bottom-up” approaches). Top-down or middle-down mass spectrometric approaches have the potential to minimize artifacts, reduce overall analysis time and provide orthogonality to this traditional approach. In this work we report a new combined approach involving middle-up LC-QTOF and middle-down LC-MALDI in-source decay (ISD) mass spectrometry. This was applied to cetuximab, panitumumab and natalizumab, selected as representative US Food and Drug Administration- and European Medicines Agency-approved mAbs. The goal was to unambiguously confirm their reference sequences and examine the general applicability of this approach. Furthermore, a new measure for assessing the integrity and validity of results from middle-down approaches is introduced – the “Sequence Validation Percentage.” Full sequence data assessment of the 3 antibodies was achieved enabling all 3 sequences to be fully validated by a combination of middle-up molecular weight determination and middle-down protein sequencing. Three errors in the reference amino acid sequence of natalizumab, causing a cumulative mass shift of only ?2 Da in the natalizumab Fd domain, were corrected as a result of this work.     

Determination of the complete amino acid sequence for the coat protein of brome mosaic virus by time-of-flight mass spectrometry. Evidence for mutations associated with change of propagation host

Time-of-flight mass spectrometry (TOFMS) has been applied to determine the complete coat protein amino acid sequences of a number of distinct brome mosaic virus (BMV) isolates. Ionization was carried out by both electrospray ionization and matrix-assisted laser desorption/ionization (MALDI). After determining overall coat protein masses, the proteins were digested with trypsin or Lys-C proteinases, and the digestion products were analyzed in a MALDI QqTOF mass spectrometer. The N terminus of the coat protein was found to be acetylated in each BMV isolate analyzed. In one isolate (BMV-Valverde), the amino acid sequence was identical to that predicted from the cDNA sequence of the "type" isolate, but deviations from the predicted amino acid sequence were observed for all the other isolates analyzed. When isolates were propagated in different host taxa, modified coat protein sequences were observed in some cases, along with the original sequence. Sequencing by TOFMS may therefore provide a basis for monitoring the effects of host passaging on a virus at the molecular level. Such TOFMS-based analyses assess the complete profiles of coat protein sequences actually present in infected tissues. They are therefore not subject to the selection biases inherent in deducing such sequences from reverse-transcribed viral RNA and cloning the resulting cDNA.     

Real time on-line amino acid analysis to explore amino acid trends and link to glycosylation outcomes of a model monoclonal antibody

Bioreactor parameters can have significant effects on the quantity and quality of biotherapeutics. Monoclonal antibody products have one particularly important critical quality attribute being the distribution of product glycoforms. N-linked glycosylation affects the therapeutic properties of the antibody including effector function, immunogenicity, stability, and clearance rate. Our past work revealed that feeding different amino acids to bioreactors altered the productivity and glycan profiles. To facilitate real-time analysis of bioreactor parameters and the glycosylation of antibody products, we developed an on-line system to pull cell-free samples directly from the bioreactors, chemically process them, and deliver them to a chromatography-mass spectroscopy system for rapid identification and quantification. We were able to successfully monitor amino acid concentration on-line within multiple reactors, evaluate glycans off-line, and extract four principal components to assess the amino acid concentration and glycosylation profile relationship. We found that about a third of the variability in the glycosylation data can be predicted from the amino acid concentration. Additionally, we determined that the third and fourth principal component accounts for 72% of our model's predictive power, with the third component indicated to be positively correlated with latent metabolic processes related to galactosylation. Here we present our work on rapid online spent media amino acid analysis and use the determined trends to collate with glycan time progression, further elucidating the correlation between bioreactor parameters such as amino acid nutrient profiles, and product quality. We believe such approaches may be useful for maximizing efficiency and reducing production costs for biotherapeutics.     

Assessment of structural and functional similarity of biosimilar products: Rituximab as a case study

Biosimilars are products that are similar in terms of quality, safety, and efficacy to an already licensed reference/ innovator product and are expected to offer improved affordability. The most significant source of reduction in the cost of development of a biosimilar is the reduced clinical examination that it is expected to undergo as compared to the innovator product. However, this clinical relief is predicated on the assumption that there is analytical similarity between the biosimilar and the innovator product. As a result, establishing analytical similarity is arguably the most important step towards successful development of a biosimilar. Here, we present results from an analytical similarity exercise that was performed with five biosimilars of rituximab (Ristova®, Roche), a chimeric mouse/ human monoclonal antibody biotherapeutic, that are available on the Indian market. The results show that, while the biosimilars exhibited similarity with respect to protein structure and function, there were significant differences with respect to size heterogeneity, charge heterogeneity and glycosylation pattern.     

In-depth structural characterization of Kadcyla® (ado-trastuzumab emtansine) and its biosimilar candidate

The biopharmaceutical industry has become increasingly focused on developing biosimilars as less expensive therapeutic products. As a consequence, the regulatory approval of 2 antibody-drug conjugates (ADCs), Kadcyla® and Adcetris® has led to the development of biosimilar versions by companies located worldwide. Because of the increased complexity of ADC samples that results from the heterogeneity of conjugation, it is imperative that close attention be paid to the critical quality attributes (CQAs) that stem from the conjugation process during ADC biosimilar development process. A combination of physicochemical, immunological, and biological methods are warranted in order to demonstrate the identity, purity, concentration, and activity (potency or strength) of ADC samples. As described here, we performed extensive characterization of a lysine conjugated ADC, ado-trastuzumab emtansine, and compared its CQAs between the reference product (Kadcyla®) and a candidate biosimilar. Primary amino acid sequences, drug-to-antibody ratios (DARs), conjugation sites and site occupancy data were acquired and compared by LC/MS methods. Furthermore, thermal stability, free drug content, and impurities were analyzed to further determine the comparability of the 2 ADCs. Finally, biological activities were compared between Kadcyla® and biosimilar ADCs using a cytotoxic activity assay and a HER2 binding assay. The in-depth characterization helps to establish product CQAs, and is vital for ADC biosimilars development to ensure their comparability with the reference product, as well as product safety.     

Physicochemical and functional assessments demonstrating analytical similarity between rituximab biosimilar HLX01 and the MabThera®

Development of bio-therapeutics has exhibited exponential growth in China over the past decade. However, no biosimilar drug has been approved in China (CN) due to the lack of a national biosimilar regulatory guidance. HLX01, a rituximab biosimilar developed in China under European Medicines Agency biosimilar guidelines and requirements, was the first such drug submitted for regulatory review in China, and it is expected to receive approval there as a biosimilar product. To demonstrate the analytical similarities of HLX01, CN-rituximab (sourced in China but manufactured in Europe) and EU-rituximab (sourced and manufactured in Europe), an extensive 3-way physicochemical and functional similarity assessment using a series of orthogonal and state-of-the-art techniques was conducted, following the similarity requirement guidelines recently published by China’s Center for Drug Evaluation. The results of the similarity study showed an identical protein amino acid sequence and highly similar primary structures between HLX01 and the reference product (RP) MabThera®, along with high similarities in higher order structures, potency, integrity, purity and impurity profiles, biological and immunological binding functions, as well as degradation behaviors under stress conditions. In addition, HLX01 presented slightly lower aggregates and better photostability compared with the RP. Despite slight changes in relative abundance of glycan moieties and heavy chain C-terminal lysine modification, no differences in biological activities and immunological properties were observed between the RP and HLX01. In conclusion, HLX01 is highly similar to CN- and EU-sourced RP in terms of physicochemical properties and biological activities, suggesting similar product quality, ef?cacy, and safety. The regulatory requirements interpreted and applied towards the HLX01 marketing application sets a precedent for analytical similarity assessment of biosimilar products in China.     

rhIL-12二硫键、N-糖基化位点及C端氨基酸序列分析

重组人白细胞介素12（rhIL-12）是一种已经用于治疗肿瘤,寄生虫、病毒性感染及造血障碍等疾病研究的异二聚体糖蛋白。结构确证是质量控制的重要内容,此研究对CHO细胞表达的rhIL-12二硫键配对方式、N-糖基化位点以及C端氨基酸序列进行了分析,使用Trypsin、Chymotrypsin和Glu-C三种酶分别对rhIL-12进行非还原酶解,尽可能地在其所有半胱氨酸残基之间断裂而形成二硫键相连的肽段,然后使用LC-MS/MS对酶解后的肽段样品进行分析,确定了rhIL-12样品中存在和理论配对方式相符的7对二硫键。将rhIL-12二硫键还原后并烷基化修饰保护,分别采用Trypsin,Chymotrypsin和GluC进行酶解,并用LC-MS/MS对酶解后肽段进行了质谱肽图及C端氨基酸序列分析,确定了rhIL-12 p35亚基C端氨基酸序列的8个氨基酸、p40亚基C端氨基酸序列的15个氨基酸。对rhIL-12样品还原及烷基化后用Trypsin变性酶解,所得肽段在H₂O及H₂¹⁸O水中分别用PNGase F糖苷酶处理酶切产物。并通过二级质谱分析脱糖后糖肽段分子量变化,从而确定了rhIL-12的3个N糖基化修饰位点,分别为p35亚基的71位和85位以及p40亚基的200位。通过建立酶解结合二级质谱鉴定的方法,证明了新药rhIL-12的二硫键位点、C端氨基酸序列和糖基化位点与理论一致。     

Seed lectin from pisum arvense: isolation, biochemical characterization and amino acid sequence

A glucose/mannose lectin was purified by affinity chromatography from Pisum arvense seeds (PAL) and the 50 kDa molecular mass in solution determined by size exclusion chromatography. SDS-PAGE and electrospray ionization mass spectrometry showed two distinct polypeptide chains: alpha (Mr. 5591 Da) and beta (19986 Da). The lectin was extensively characterized in terms of its biochemical and biological aspects. The amino acid sequence was established by Edman degradation of overlapping peptides. PAL in solution behaves as a dimer and has its monomeric structure formed by two distinct polypeptide chains named alpha (Mr. 5591 Da) and beta (19986 Da) by Electrospray ionization (ESI) mass spectrometry. PAL possesses identical amino acid sequences to that of pea seed lectin but undoubtedly does not exhibit sequence heterogeneity. It is discussed that P. arvense should be considered as a synonym of P. sativum. Furthermore, like pea lectin, PAL discriminates biantennary fucosylated glycan, determined by surface plasmon resonance.     

The amino-acid sequence of isoinhibitor K form snails (Helix pomatia). A sequence determination by automated Edman degradation and mass-spectral identification of the phenylthiohydantoins.

Performic-acid-oxidized isoinhibitor K of snails (Helix pomatia) was subjected to arginine-directed tryptic proteolysis. Six peptide fragments including one overlap peptide from limited cleavage of the Arg-3-Pro-4 bond were purified to homogeneity. Four arginine peptides and the C-terminal peptide were sequenced by automatic Edman degradation using a special peptide program. The phenylthiohydantoins were all identified by chemical ionization mass spectrometry, except four cysteic acid residues that were identified on an amino acid analyzer after acid hydrolysis. Quantitative evaluation of the phenylthiohydantoins by chemical ionization mass spectrometry using total molecular-ion beam integration greatly facilitated sequencing. The mass spectrum of the dipeptide less than Glu-Gly revealed that the N-terminus was blocked by pyroglutamic acid. The complete amino acid sequence of isoinhibitor K was determined. An almost 50% homology between the sequences of the snail inhibitor and the bovine trypsin-kallikrein inhibitor (Kunitz) became obvious. A comparison of all homologous sequences of this particular class of proteins known to date is presented.     

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.     

Identification of disulfide bonds and site-specific glycosylation in chicken alpha1-acid glycoprotein by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Recently, we reported the amino acid sequence of chicken alpha1-acid glycoprotein (chicken alpha1-AGP) [Biochem. Biophys. Res. Commun. 295 (2002) 587]. In this study, we located the disulfide bonds and site-specific glycosylation in chicken alpha1-AGP using tryptic digests of carbamidomethylated chicken alpha1-AGP, carbamidomethylated completely deglycosylated chicken alpha1-AGP (cd-alpha1-AGP), and nonreduced denatured cd-alpha1-AGP by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Based on the detection of peptides mlz 3037.4 (amino acid sequences 69-76 plus 161-183) and 3453.3 (amino acid sequences 69-80 plus 161-183), the two disulfide bonds of chicken alpha1-AGP were determined to be located at Cys 6-Cys 146 and Cys 73-Cys 163. The results also showed that Asn 16, 70, 77, and 87 were fully glycosylated and that Asn 62 was partially glycosylated.     

一种优化的MALDI-TOF质谱分析多肽C端序列方法

利用基质辅助激光解吸飞行时间 (MALDI TOF)质谱技术 ,测定羧肽酶Y消化蛋白质和多肽 .所产生的缩短肽片段的质量 ,在一张谱图上得到各个不同酶解时间所形成的肽质量梯度 .根据谱图中相邻两肽峰的质量差得到切去氨基酸的信息 ,从而读出C端氨基酸序列 .在pmol水平下对人促肾上腺皮质激素片段 (ACTH 1 3 9) ,人血管紧张肽片段 (angiotensin Ⅰ ,angiotensin Ⅱ )的C端序列进行了测定 .讨论了在不同浓度 ,不同时间 ,不同温度下酶解所得到的序列测定结果 .在优化条件下 ,人ACTH片段得到了C端 2 0个氨基酸残基顺序 ,为目前C端序列分析所得到的最长序列     

Mass spectrometric-based revision of the structure of a cysteine-rich peptide toxin with gamma-carboxyglutamic acid, TxVIIA, from the sea snail, Conus textile.

A mollusk-specific toxin, TxVIIA, having potent paralytic activity was isolated from the venom of sea snail Conus textile (Fainzilber M et al., 1991, Eur J Biochem 202:589-595). The structure reported above was based upon amino acid analysis and the Edman degradation. We have recently reinvestigated this toxin employing some of the most novel techniques in mass spectrometry. We now report a revised structure based primarily on high-energy collision-induced dissociation analysis of the two Asp17-N peptides of the reduced, pyridinylethyl derivative representing the entire sequence using matrix-assisted laser desorption ionization (MALDI) as CGGYSTYC gamma VDS gamma CCSDNCVRSYCTLF-NH2 (gamma, gamma-carboxyglutamic acid or Gla). The N-terminus of the previous sequence was incorrect, apparently due to a side reaction of reduction and alkylation, which led to the erroneous assignment of Trp for the N-terminal residue. In addition, the last two C-terminal amino acids and the C-terminal amidation had not been detected. Also, a combination of electrospray ionization mass spectrometry and positive and negative ion MALDI mass spectrometry provided information on the molecular weights of the native and derivatized toxin and presence of two Gla residues. Thus, TxVIIA does not have an "unusual" sequence as previously reported, but in fact belongs to the conserved Cys framework for omega- and delta-conotoxins. However, the four net negative charges with the cysteine-rich structure of this revised sequence is highly unusual for conopeptides.