RESUMO
Monoclonal antibodies (mAbs) have become predominant therapeutics by providing highly specific mechanisms of action enabling treatment of complex diseases. However, mAbs themselves are highly complex and require thorough testing and characterization to ensure efficacy and patient safety. In this regard, fragmentation is a degradation product of concern. The biotechnology industry uses capillary gel electrophoresis (CGE) to quantify fragmentation by electrophoretically resolving size variants, such as products resulting from partial reduction of interchain disulfides. However, standard CGE methods may not adequately separate less typical fragments, particularly when there is minimal size difference to the parent molecule. For mAb-1, a degradant only â¼11 kDa smaller than the intact mAb (â¼149 kDa) was unable to be resolved under typical non-reducing conditions, preventing an accurate purity assessment and precluding tracking of product purity within stability studies. To address these deficiencies, a subunit-based non-reducing CGE method was developed to employ IdeS protease to produce F(ab')2 and Fc fragments, which resulted in baseline resolution of the clipped subunit species from its parent species. This enabled more accurate trending of purity throughout stability studies. Method characterization ensured that this subunit method monitored expected impurities observed by intact non-reducing CGE and thus could suitably replace non-reducing CGE in the release and stability testing panel. It also has the potential to replace reducing CGE based on its tracking of the deglycosylated Fc species. We believe this approach of utilizing proteases to develop subunit CGE methods for release and stability can be applied to other molecules when in need of resolving analogous fragments.
Assuntos
Anticorpos Monoclonais , Peptídeo Hidrolases , Humanos , Anticorpos Monoclonais/análise , Eletroforese Capilar/métodos , Imunossupressores , Endopeptidases , DigestãoRESUMO
Liquid chromatography-mass spectrometry (LC-MS)-based proteomics approaches have been widely used to identify residual host-cell proteins (HCPs) in support of process and product characterization for protein therapeutics. Particularly, these methods can provide a general and unbiased approach for the detection of HCPs and may generate critical information on HCPs that are outside the coverage provided by a conventional immunoassay. A significant technical hurdle for HCP analysis is the overwhelmingly large background of biotherapeutic that obscures HCP detection and quantification. In this work, we developed a method that relies on hydrophilic interaction chromatography (HILIC) for HCP enrichment followed by in situ concentration and digestion prior to LC-MS analysis. This approach has enabled detection of HCPs in a drug substance that were not observed in other conventional flow rate LC-MS strategies. For example, 28% of HCPs identified in NISTmAb (20 out of 71) were not previously published or identified by established methods such as the native digestion technique. For an IgG1 protein spiked with 1000 ppm HCP standards, we detected 83 HCPs, 61 out of which were not identified by the native digestion method. Similar improvement in performance was demonstrated for an Fc-fusion protein therapeutic. Our method can be readily implemented in most protein mass spectrometry laboratories to support process development for protein therapeutics.
Assuntos
Cromatografia Líquida/métodos , Proteínas/química , Proteínas/uso terapêutico , Animais , Anticorpos Monoclonais , Anticorpos Monoclonais Humanizados , Células CHO , Cricetulus , Imunoglobulina G/química , Espectrometria de Massas em Tandem/métodos , Tripsina/metabolismoRESUMO
RATIONALE: Multi-Attribute Methods (MAMs) are appealing due to their ability to provide data on multiple molecular attributes from a single assay. If fully realized, such tests could reduce the number of assays required to support a product control strategy while providing equivalent or greater product understanding relative to the conventional approach. In doing so, MAMs have the potential to decrease development and manufacturing costs by reducing the number of tests in a release panel. METHODS: In this work, we report a MAM which is based on subunit mass analysis. RESULTS: The MAM assay is shown to be suitable for use as a combined method for identity testing, glycan profiling, and protein ratio determination for co-formulated monoclonal antibody (mAb) drugs. This is achieved by taking advantage of the high mass accuracy and relative quantification capabilities of intact mass analysis using quadrupole time-of-flight mass spectrometry (Q-TOF MS). Protein identification is achieved by comparing the measured masses of light chain (LC) and heavy chain (HC) mAbs against their theoretical values. Specificity is based on instrument mass accuracy. Glycan profiling and relative protein ratios are determined by the relative peak intensities of the protein HC glycoforms and LC glycoforms, respectively. Results for these relative quantifications agree well with those obtained by the conventional hydrophilic interaction liquid chromatography (HILIC) and reversed-phase LC methods. CONCLUSIONS: The suitability of this MAM for use in a quality control setting is demonstrated through assessment specificity for mAb identity, and accuracy, precision, linearity and robustness for glycan profiling and ratio determination. Results from this study indicate that a MAM with subunit mass analysis has the potential to replace three conventional methods widely used for mAb release testing including identification assay, glycosylation profiling, and ratio determination for co-formulated mAbs.
Assuntos
Anticorpos Monoclonais/análise , Anticorpos Monoclonais/química , Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Animais , Anticorpos Monoclonais/metabolismo , Células CHO , Cricetulus , Glicosilação , Humanos , Espectrometria de Massas/instrumentação , Polissacarídeos/análise , Subunidades Proteicas/análise , Subunidades Proteicas/química , Proteínas/análise , Proteínas Recombinantes/análise , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sensibilidade e EspecificidadeRESUMO
Process control for manufacturing biologics is critical for ensuring product quality, safety, and lot to lot consistency of therapeutic proteins. In this study, we investigated the root cause of the pink coloration observed for various in-process pools and drug substances in the antibody manufacturing process. Vitamin B12 is covalently bound to mAbs via a cobalt-sulfur coordinate bond via the cysteine residues. The vitamin B12 was identified to attach to an IgG4 molecule at cysteine residues on light chain (Cys-214), and heavy chain (Cys-134, Cys-321, Cys-367, and Cys-425). Prior to attachment to mAbs, the vitamin B12 needs to be in its active form of hydroxocobalamin. During culture media preparation, storage and cell culture processing, cyanocobalamin, the chemical form of vitamin B12 added to media, is converted to hydroxocobalamin by white fluorescence light (about 50% degradation in 11-14 days at room temperature and with room light intensity about 500-1,000 lux) and by short-wavelength visible light (400-550 nm). However, cyanocobalamin is stable under red light (wavelength >600 nm) exposure and does not convert to hydroxocobalamin. Our findings suggests that the intensity of pink color depends on concentrations of both free sulfhydryl groups on reduced mAb and hydroxocobalamin, the active form of vitamin B12 . Both reactants are necessary and neither one of them is sufficient to generate pink color, therefore process control strategy can consider limiting either one or both factors. A process control strategy to install red light (wavelength >600 nm) in culture media preparation, storage and culture processing areas is proposed to provide safe light for biologics and to prevent light-induced color variations in final products.
Assuntos
Anticorpos Monoclonais/química , Hidroxocobalamina/química , Imunoglobulina G/química , Vitamina B 12/química , Anticorpos Monoclonais/análise , Produtos Biológicos/análise , Produtos Biológicos/química , Cobalto/análise , Cobalto/química , Qualidade de Produtos para o Consumidor , Meios de Cultura/análise , Meios de Cultura/química , Cisteína/análise , Cisteína/química , Dissulfetos/análise , Dissulfetos/química , Humanos , Hidroxocobalamina/análise , Imunoglobulina G/análise , Luz , Vitamina B 12/análiseRESUMO
This study reports findings of an unusual cluster of mutations spanning 22 bp (base pairs) in a monoclonal antibody expression vector. It was identified by two orthogonal methods: mass spectrometry on expressed protein and next-generation sequencing (NGS) on the plasmid DNA. While the initial NGS analysis confirmed the designed sequence modification, intact mass analysis detected an additional mass of the antibody molecule expressed in CHO cells. The extra mass was eventually found to be associated with unmatched nucleotides in a distal region by checking full-length sequence alignment plots. Interestingly, the complementary sequence of the mutated sequence was a reverse sequence of the original sequence and flanked by two 10-bp reverse-complementary sequences, leading to an undesirable DNA recombination. The finding highlights the necessity of rigorous examination of expression vector design and early monitoring of molecule integrity at both DNA and protein levels to prevent clones from having sequence variants during cell line development.
Assuntos
Anticorpos/metabolismo , Vetores Genéticos , Fatores Imunológicos/metabolismo , Mutação , Proteínas Recombinantes/metabolismo , Animais , Anticorpos/química , Anticorpos/genética , Células CHO , Cricetulus , Sequenciamento de Nucleotídeos em Larga Escala , Fatores Imunológicos/química , Fatores Imunológicos/genética , Espectrometria de Massas , Plasmídeos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Recombinação GenéticaRESUMO
PURPOSE: Tryptophan's (Trp) unique hydrophobic and structural properties make it an important antigen binding motif when positioned in complementarity-determining regions (CDRs) of monoclonal antibodies (mAbs). Oxidation of Trp residues within the CDR can deleteriously impact antigen binding, particularly if the CDR conformation is altered. The goal of this study was to evaluate the conformational and functional impact of Trp oxidation for two mAb subtypes, which is essential in determining the structure-function relationship and establishing appropriate analytical control strategies during protein therapeutics development. METHODS: Selective Trp oxidation was induced by 2,2'-Azobis(2-amidinopropane) dihydrochloride (AAPH) treatment in the presence of free methionine (Met). The native and chemically oxidized mAbs were characterized by hydrogen-deuterium exchange mass spectrometry (HDX-MS) for conformational changes and surface plasmon resonance (SPR) for antigen-antibody binding. RESULTS: Treatment of mAbs with AAPH selectively oxidized solvent accessible Trp residues. Oxidation of Trp within or in proximity of CDRs increased conformational flexibility in variable domains and disrupted antigen binding. CONCLUSIONS: Trp oxidation in CDRs can adversely impact mAbs' conformation and antigen binding. Trp oxidation should be carefully evaluated as part of critical quality attribute assessments. Oxidation susceptible Trp should be closely monitored during process development for mAbs to establish appropriate analytical control for manufacturing of drug substance and drug product.
Assuntos
Anticorpos Monoclonais/química , Regiões Determinantes de Complementaridade/química , Deutério/química , Hidrogênio/química , Triptofano/química , Antígenos/química , Medição da Troca de Deutério/métodos , Espectrometria de Massas/métodos , Oxirredução , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície/métodosRESUMO
Recombinant monoclonal antibodies (mAbs) manufactured from immortalized mammalian cell lines are becoming increasingly important as therapies. Ensuring the quality of expressed proteins is critical when developing manufacturing processes. Protein sequence variants (PSVs) are a type of product-related variant in which errors in the protein sequence are present. Detecting PSVs and determining their origins, either by DNA mutation or mRNA mistranslation, is critical. Mutations cannot be remediated without developing new clones, which can be costly and time-consuming. In contrast, mistranslation can usually be mitigated by optimizing cell culture conditions. In this work, we first developed a new method to detect low-abundance PSVs with improved sensitivity. Then, a statistical metric was proposed to determine whether the observed PSVs originate from mutation or mistranslation by characterizing the distribution of PSVs. This method was applied to the evaluation of 50 clones from five mAbs programs, allowing for identification of five mutation and 139 mistranslation PSVs. The presence of even a few mutations demonstrates the necessity of clone screening during process development.
Assuntos
Anticorpos Monoclonais/análise , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Animais , Anticorpos Monoclonais/genética , Células CHO , Códon/genética , Cricetulus , Mutação , Biossíntese de Proteínas/genéticaRESUMO
Chemical modifications can potentially change monoclonal antibody's (mAb) local or global conformation and therefore impact their efficacy as therapeutic drugs. Modifications in the complementarity-determining regions (CDRs) are especially important because they can impair the binding affinity of an antibody for its target and therefore drug potency as a result. In order to understand the impact on mAb attributes induced by specific chemical modifications within the CDR, hydrogen-deuterium exchange mass spectrometry (HDX MS) was used to interrogate the conformational impact of Asp isomerization and Met oxidation in the CDRs of a model monoclonal antibody (mAb1). Our results indicate that despite their proximity to each other, Asp54 isomerization and Met56 oxidation in CDR2 in the heavy chain of mAb1 result in opposing conformational impacts on the local and nearby regions, leading directly to different alterations on antibody-antigen binding affinity. This study revealed direct evidence of local and global conformational changes caused by two of the most common degradation pathways in the CDRs of a mAb and identified correlations between chemical modification, structure, and function of the therapeutic monoclonal antibody.
Assuntos
Anticorpos Monoclonais/química , Medição da Troca de Deutério , Espectrometria de Massas , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Afinidade de Anticorpos , Reações Antígeno-Anticorpo , Ácido Aspártico/química , Células CHO , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/metabolismo , Cricetinae , Cricetulus , Deutério/química , Ensaio de Imunoadsorção Enzimática , Hidrogênio/química , Isomerismo , Cinética , Metionina/química , Oxirredução , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
PURPOSE: Discoloration of protein therapeutics has drawn increased attention recently due to concerns of potential impact on quality and safety. Investigation of discoloration in protein therapeutics for comparability is particularly challenging primarily for two reasons. First, the description of color or discoloration is to certain extent a subjective characteristic rather than a quantitative attribute. Secondly, the species contributing to discoloration may arise from multiple sources and are typically present at trace levels. Our purpose is to development a systematic approach that allows effective identification of the color generating species in protein therapeutics. METHODS: A yellow-brown discoloration event observed in a therapeutic protein was investigated by optical spectroscopy, ultra-performance liquid chromatography, and mass spectrometry (MS). RESULTS: Majority of the color generating species were identified as oxidatively modified protein. The location of the oxidized amino acid residues were identified by MS/MS. In addition, the impact of process-related impurities co-purified from media on discoloration was also investigated. Finally a semi-quantitative scale to estimate the contribution of each color source is presented, which revealed oxidized peptides are the major contributors. CONCLUSIONS: A systematic approach was developed for identification of the color generating species in protein therapeutics and for estimation of the contribution of each color source.
Assuntos
Peptídeos/química , Proteínas/química , Animais , Células CHO , Cromatografia Líquida de Alta Pressão/métodos , Cor , Cricetulus , Contaminação de Medicamentos , Histidina/análise , Oxirredução , Preparações Farmacêuticas/química , Proteínas Recombinantes de Fusão/química , Espectrometria de Massas em Tandem/métodos , Triptofano/análiseRESUMO
The multi-attribute method (MAM) was conceived as a single assay to potentially replace multiple single-attribute assays that have long been used in process development and quality control (QC) for protein therapeutics. MAM is rooted in traditional peptide mapping methods; it leverages mass spectrometry (MS) detection for confident identification and quantitation of many types of protein attributes that may be targeted for monitoring. While MAM has been widely explored across the industry, it has yet to gain a strong foothold within QC laboratories as a replacement method for established orthogonal platforms. Members of the MAM consortium recently undertook an interlaboratory study to evaluate the industry-wide status of MAM. Here we present the results of this study as they pertain to the targeted attribute analytics component of MAM, including investigation into the sources of variability between laboratories and comparison of MAM data to orthogonal methods. These results are made available with an eye toward aiding the community in further optimizing the method to enable its more frequent use in the QC environment.
Assuntos
Benchmarking , Proteínas , Espectrometria de Massas/métodos , Mapeamento de Peptídeos/métodos , Controle de QualidadeRESUMO
The Multi-Attribute Method (MAM) Consortium was initially formed as a venue to harmonize best practices, share experiences, and generate innovative methodologies to facilitate widespread integration of the MAM platform, which is an emerging ultra-high-performance liquid chromatography-mass spectrometry application. Successful implementation of MAM as a purity-indicating assay requires new peak detection (NPD) of potential process- and/or product-related impurities. The NPD interlaboratory study described herein was carried out by the MAM Consortium to report on the industry-wide performance of NPD using predigested samples of the NISTmAb Reference Material 8671. Results from 28 participating laboratories show that the NPD parameters being utilized across the industry are representative of high-resolution MS performance capabilities. Certain elements of NPD, including common sources of variability in the number of new peaks detected, that are critical to the performance of the purity function of MAM were identified in this study and are reported here as a means to further refine the methodology and accelerate adoption into manufacturer-specific protein therapeutic product life cycles.
RESUMO
Isomerization of surface-exposed aspartic acid (Asp) in the complementarity-determining regions of therapeutic proteins could potentially impact their target binding affinity because of the sensitive location, and often requires complex analytical tactics to understand its effect on structure-function and stability. Inaccurate quantitation of Asp-isomerized variants, especially the succinimide intermediate, presents major challenge in understanding Asp degradation kinetics, its stability, and consequently establishing a robust control strategy. As a practical solution to this problem, a comprehensive analytical tool kit has been developed, which provides a solution to fully characterize and accurately quantify the Asp-related product variants. The toolkit offers a combination of 2 steps, an ion-exchange chromatography method to separate and enrich the isomerized variants in the folded structure for structure-function evaluation and a novel focused peptide mapping method to quantify the individual complementarity-determining region isomerization components including the unmodified Asp, succinimide, and isoaspartate. This novel procedure allowed an accurate quantification of each Asp-related variant and a comprehensive assessment of the functional impact of Asp isomerization, which ultimately helped to establish an appropriate control strategy for this critical quality attribute.
Assuntos
Anticorpos Monoclonais/química , Ácido Aspártico/química , Regiões Determinantes de Complementaridade/química , Ensaios de Triagem em Larga Escala/métodos , Imunoglobulina G/química , Mapeamento de Peptídeos/métodos , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia por Troca Iônica/métodos , Composição de Medicamentos , Estabilidade de Medicamentos , Armazenamento de Medicamentos , Fragmentos Fab das Imunoglobulinas/química , Ácido Isoaspártico/química , Isomerismo , Estabilidade Proteica , Relação Estrutura-Atividade , Succinimidas/química , Espectrometria de Massas em Tandem/métodosRESUMO
During large-scale monoclonal antibody manufacturing, disulfide bond reduction of antibodies, which results in generation of low molecule weight species, is occasionally observed. When this happens, the drug substance does not meet specifications. Many investigations have been conducted across the biopharmaceutical industry to identify the root causes, and multiple strategies have been proposed to mitigate the problem. The reduction is correlated with the release of cellular reducing components and depletion of dissolved oxygen before, during, and after harvest. Consequently, these factors can lead to disulfide reduction over long-duration storage at room temperature prior to Protein A chromatography. Several strategies have been developed to minimize antibody reduction, including chemical inhibition of reducing components, maintaining aeration before and after harvest, and chilling clarified harvest during holding. Here, we explore the use of hydrogen peroxide in clarified harvest bulk or cell culture fluid as a strategy to prevent disulfide reduction. A lab-scale study was performed to demonstrate the effectiveness of hydrogen peroxide in preventing antibody reduction using multiple IgG molecules. Studies were done to define the optimal concentration of hydrogen peroxide needed to avoid unnecessary oxidization of the antibody products. We show that adding a controlled amount of hydrogen peroxide does not change product quality attributes of the protein. Since hydrogen peroxide is soluble in aqueous solutions and decomposes into water and oxygen, there is no additional burden involved in removing it during the downstream purification steps. Due to its ease of use and minimal product impact, we demonstrate that hydrogen peroxide treatment is a powerful, simple tool to quench reducing potential by simply mixing it with harvested cell culture fluid.
Assuntos
Anticorpos Monoclonais , Dissulfetos/química , Peróxido de Hidrogênio/química , Imunoglobulina G , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Células CHO , Cromatografia de Afinidade/métodos , Cricetulus , Imunoglobulina G/química , Imunoglobulina G/isolamento & purificação , Oxirredução , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteína Estafilocócica A/químicaRESUMO
Protein higher order structure (HOS) is an essential quality attribute to ensure protein stability and proper biological function. Protein HOS characterization is performed during comparability assessments for product consistency as well as during forced degradation studies for structural alteration upon stress. Circular dichroism (CD) spectroscopy is a widely used technique for measuring protein HOS, but it remains difficult to assess HOS with a high degree of accuracy and precision. Moreover, once spectral changes are detected, interpreting the differences in terms of specific structural attributes is challenging. Spectral normalization by the protein concentration remains one of the largest sources of error and reduces the ability to confidently detect differences in CD spectra. This work develops a simple method to enhance the precision of the CD spectral measurements through normalization of the CD spectra by the protein concentration determined directly from the CD measurement. This method is implemented to successfully detect small CD spectral changes in multiple forced degradation studies as well as comparability assessments during biologics drug development. Furthermore, the interpretation of CD spectral changes in terms of HOS differences are provided based on orthogonal data in conjunction with structural insights gained through in silico homology modeling of the protein structure.
Assuntos
Produtos Biológicos/química , Proteínas/química , Dicroísmo Circular/métodos , Conformação ProteicaRESUMO
Variants of monoclonal antibody containing an extra light chain have been reported in protein products. Due to potential impact on potency and immunogenicity, it is important to understand the formation mechanism of such variants so that appropriate control strategies can be implemented to assure product quality. In a model monoclonal antibody, we observed a size variant with an extra light chain noncovalently associated with the monomer (later named as "1.2mer"). The interaction between monomer and the extra light chain was characterized by native spray and hydrogen-deuterium exchange mass spectrometry techniques. The goal is to understand the nature of the noncovalent interaction, to map out the interaction interface and regions of potential conformational distortions. In addition, computational modeling was used to aid in binding site identification. The combined results identify the interaction interface to be located in the heavy chain region 38-57 and in the extra light chain region 30-50. To the best of our knowledge, this study is the first to characterize noncovalent interaction of a size variant comprising an antibody monomer and an extra light chain. Structural knowledge generated in this research work is invaluable for process development and construct design of antibody-based biopharmaceuticals.
Assuntos
Anticorpos Monoclonais/química , Imunoglobulina G/química , Espectrometria de Massas/métodos , Animais , Sítios de Ligação de Anticorpos , Células CHO , Cromatografia em Gel , Cricetulus , Deutério/análise , Medição da Troca de Deutério/métodos , Humanos , Hidrogênio/análise , Modelos Moleculares , Proteínas Recombinantes/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Homologia Estrutural de ProteínaRESUMO
Sequence variants of a monoclonal antibody resulting from incomplete processing of signal peptide were identified and characterized using multiple mass spectrometry platforms and reverse phase chromatography. Detection and quantification of these variants by three LC/MS platforms were assessed. Quantification was also performed by mass spectrometric analysis of the subunits of the antibody generated by reduction and IdeS proteolysis. Peptide mapping with LC/MS/MS detection was used to quantify and confirm the identities of signal peptide sequence variants. Although quantification of the signal peptide variants thru mass spectrometry approaches is system dependent, our data revealed the results are close to the values determined by chromatographic separation with UV detection. Each of the methods have proven effective in demonstrating the consistency of signal peptide variants levels across the manufacture history of the antibody.
Assuntos
Anticorpos Monoclonais/análise , Anticorpos Monoclonais/química , Imunoglobulina G/análise , Imunoglobulina G/química , Sinais Direcionadores de Proteínas , Animais , Linhagem Celular , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia de Fase Reversa , Peso Molecular , Mapeamento de Peptídeos , Espectrometria de Massas em Tandem/métodosRESUMO
Low levels of alanine to serine sequence variants were identified in an IgG4 monoclonal antibody by ultra/high performance liquid chromatography and tandem mass spectrometry. The levels of the identified sequence variants A183S and A152S, both in the light chain, have been determined to be 7.8-9.9% and 0.5-0.6%, by extracted ion currents of the tryptic peptides L16 and L14, respectively. The A183S variant was confirmed through tryptic map spiking experiments using synthetic peptide, SDYEK, which incorporated Ser at the position of native Ala in the tryptic peptide L16. Both mutations were also observed by endoproteinase Asp-N peptide mapping. The variant level of A183S was also quantified by LC-UV with detection at 280nm and fluorescence detection of tyrosine residues on the tryptic peptides. The results from LC-MS, UV, and fluorescence detection are in close agreement with each other. The levels of the sequence variants are comparable among the antibody samples manufactured at different scales as well as locations, indicating that the variants' levels are not affected by manufacture scale or locations. DNA sequencing of the master cell bank revealed the presence of mixed bases at position 183 encoding both wild and mutated populations, whereas bases encoding the minor sequence variant at position 152 were not detected. The root cause for A152S mutation is not yet clearly understood at this moment.
Assuntos
Alanina/química , Anticorpos Monoclonais/química , Imunoglobulina G/química , Mapeamento de Peptídeos/métodos , Serina/química , Alanina/análise , Alanina/genética , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/genética , Células CHO , Cromatografia Líquida de Alta Pressão/métodos , Cricetinae , Cricetulus , DNA Complementar/genética , Humanos , Imunoglobulina G/genética , Dados de Sequência Molecular , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/química , Serina/análise , Serina/genética , Espectrometria de Massas em Tandem/métodos , Tripsina/químicaRESUMO
A novel amino acid misincorporation, in which the intended glycine (Gly) residues were replaced by a glutamic acid (Glu), was observed in a recombinant protein expressed by Escherichia coli. The misincorporation was identified by peptide mapping and liquid chromatography-tandem mass spectrometric analysis on proteolyzed peptides of the protein and verified using the corresponding synthetic peptides containing the misincorporated residues. Analysis of the distribution of the misincorporated residues and their codon usage shows strong correlation between this misincorporation and the use of rarely used codon within the E. coli expression system. Results in this study suggest that the usage of the rare codon GGA has resulted in a Glu for Gly misincorporation.
Assuntos
Códon , Escherichia coli/genética , Ácido Glutâmico/genética , Glicina/genética , Proteínas Recombinantes/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Escherichia coli/metabolismo , Ácido Glutâmico/química , Ácido Glutâmico/metabolismo , Glicina/química , Glicina/metabolismo , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Liquid chromatography mass spectrometry (LC-MS) peptide mapping can be a versatile technique for characterizing protein glycosylation sites without the need to remove the attached glycans as in conventional oligosaccharide mapping methods. In this way, both N-linked and O-linked sites of glycosylation can each be directly identified, characterized, and quantified by LC-MS as intact glycopeptides in a single experiment. LC-MS peptide mapping of the individual glycosylation sites avoids many of the limitations of preparing and analyzing an entire pool of released N-linked oligosaccharides from all sites mixed together. In this study, LC interfaced to a linear ion trap mass spectrometer (ESI-LIT-MS) were used to characterize the glycosylation of a recombinant IgG1 monoclonal antibody and a CTLA4-Ig fusion protein with multiple sites of N-and O-glycosylation. Samples were reduced, S-carboxyamidomethylated, and cleaved with either trypsin or endoproteinase Asp-N. Enhanced detection for minor IgG1 glycoforms (â¼0.1 to 1.0 mol% level) was obtained by LC-MS of the longer 32-residue Asp-N glycopeptide (4+ protonated ion) compared to the 9-residue tryptic glycopeptide (2+ ion). LC-MS peptide mapping was run according to a general procedure: (1) Locate N-linked and/or O-linked sites of glycosylation by selected-ion-monitoring of carbohydrate oxonium fragment ions generated by ESI in-source collision-induced dissociation (CID), i.e. 204, 366, and 292 Da marker ions for HexNAc, HexNAc-Hex, and NeuAc, respectively; (2) Characterize oligosaccharides at each site via MS and MSMS. Use selected ion currents (SIC) to estimate relative amounts of each glycoform; and (3) Measure the percentage of site-occupancy by searching for any corresponding nonglycosylated peptide.