“…The resulting levels (as well as standard deviations) of in-sample oxidation in the peptides containing Met sulfoxide are in good agreement with the similar manual analysis reported by an interlaboratory study on identification and quantification of the NISTmAb methionine oxidation ( Figure 8 ). 36 Consistent with that study, Met 255 and Met 431 are the two most oxidized sites, with 3.4% and 3.0% abundance relative to the corresponding unoxidized sites, respectively. Next are Met 34 of the heavy chain and Met 32 of the light chain, with approximately 2% oxidation.…”
Section: Applications Of the Nistmab Spectral Library To Structure Ansupporting
confidence: 87%
“…Met 361, Met 87 and Met 4 exhibit low levels of observed oxidation, at 0.7%, 1.3%, and 1.4%, respectively. As demonstrated in both studies of the NISTmAb, 36 accurate methionine quantification requires careful separation of native and artifact oxidized peptides. Figure 8.…”
Section: Applications Of the Nistmab Spectral Library To Structure Anmentioning
confidence: 99%
“… Comparison of relative abundance of each oxidized Met reside in the NISTmAb reported by this study and an interlaboratory study of identification and quantification of NISTmAb methionine oxidation. 36 NIST, this study; LAB1 – LAB3, Laboratory 1 to Laboratory 3 in Reference 36. LAB3 conducted a manual quantification of methionine oxidation.…”
Section: Applications Of the Nistmab Spectral Library To Structure Anmentioning
We describe the creation of a mass spectral library composed of all identifiable spectra derived from the tryptic digest of the NISTmAb IgG1κ. The library is a unique reference spectral collection developed from over six million peptide-spectrum matches acquired by liquid chromatography-mass spectrometry (LC-MS) over a wide range of collision energy. Conventional one-dimensional (1D) LC-MS was used for various digestion conditions and 20- and 24-fraction two-dimensional (2D) LC-MS studies permitted in-depth analyses of single digests. Computer methods were developed for automated analysis of LC-MS isotopic clusters to determine the attributes for all ions detected in the 1D and 2D studies. The library contains a selection of over 12,600 high-quality tandem spectra of more than 3,300 peptide ions identified and validated by accurate mass, differential elution pattern, and expected peptide classes in peptide map experiments. These include a variety of biologically modified peptide spectra involving glycosylated, oxidized, deamidated, glycated, and N/C-terminal modified peptides, as well as artifacts. A complete glycation profile was obtained for the NISTmAb with spectra for 58% and 100% of all possible glycation sites in the heavy and light chains, respectively. The site-specific quantification of methionine oxidation in the protein is described. The utility of this reference library is demonstrated by the analysis of a commercial monoclonal antibody (adalimumab, Humira®), where 691 peptide ion spectra are identifiable in the constant regions, accounting for 60% coverage for both heavy and light chains. The NIST reference library platform may be used as a tool for facile identification of the primary sequence and post-translational modifications, as well as the recognition of LC-MS method-induced artifacts for human and recombinant IgG antibodies. Its development also provides a general method for creating comprehensive peptide libraries of individual proteins.
“…The resulting levels (as well as standard deviations) of in-sample oxidation in the peptides containing Met sulfoxide are in good agreement with the similar manual analysis reported by an interlaboratory study on identification and quantification of the NISTmAb methionine oxidation ( Figure 8 ). 36 Consistent with that study, Met 255 and Met 431 are the two most oxidized sites, with 3.4% and 3.0% abundance relative to the corresponding unoxidized sites, respectively. Next are Met 34 of the heavy chain and Met 32 of the light chain, with approximately 2% oxidation.…”
Section: Applications Of the Nistmab Spectral Library To Structure Ansupporting
confidence: 87%
“…Met 361, Met 87 and Met 4 exhibit low levels of observed oxidation, at 0.7%, 1.3%, and 1.4%, respectively. As demonstrated in both studies of the NISTmAb, 36 accurate methionine quantification requires careful separation of native and artifact oxidized peptides. Figure 8.…”
Section: Applications Of the Nistmab Spectral Library To Structure Anmentioning
confidence: 99%
“… Comparison of relative abundance of each oxidized Met reside in the NISTmAb reported by this study and an interlaboratory study of identification and quantification of NISTmAb methionine oxidation. 36 NIST, this study; LAB1 – LAB3, Laboratory 1 to Laboratory 3 in Reference 36. LAB3 conducted a manual quantification of methionine oxidation.…”
Section: Applications Of the Nistmab Spectral Library To Structure Anmentioning
We describe the creation of a mass spectral library composed of all identifiable spectra derived from the tryptic digest of the NISTmAb IgG1κ. The library is a unique reference spectral collection developed from over six million peptide-spectrum matches acquired by liquid chromatography-mass spectrometry (LC-MS) over a wide range of collision energy. Conventional one-dimensional (1D) LC-MS was used for various digestion conditions and 20- and 24-fraction two-dimensional (2D) LC-MS studies permitted in-depth analyses of single digests. Computer methods were developed for automated analysis of LC-MS isotopic clusters to determine the attributes for all ions detected in the 1D and 2D studies. The library contains a selection of over 12,600 high-quality tandem spectra of more than 3,300 peptide ions identified and validated by accurate mass, differential elution pattern, and expected peptide classes in peptide map experiments. These include a variety of biologically modified peptide spectra involving glycosylated, oxidized, deamidated, glycated, and N/C-terminal modified peptides, as well as artifacts. A complete glycation profile was obtained for the NISTmAb with spectra for 58% and 100% of all possible glycation sites in the heavy and light chains, respectively. The site-specific quantification of methionine oxidation in the protein is described. The utility of this reference library is demonstrated by the analysis of a commercial monoclonal antibody (adalimumab, Humira®), where 691 peptide ion spectra are identifiable in the constant regions, accounting for 60% coverage for both heavy and light chains. The NIST reference library platform may be used as a tool for facile identification of the primary sequence and post-translational modifications, as well as the recognition of LC-MS method-induced artifacts for human and recombinant IgG antibodies. Its development also provides a general method for creating comprehensive peptide libraries of individual proteins.
“…Most common PTMs in this class are glycosylation, disulfide bond formation, and proteolytic cleavage of the protein. Chemical modifications are generated during upstream and downstream processing, formulation, and storage, including oxidation, deamidation, isomerization, glycation, and Gln/Glu cyclization [80]. Those PTMs can affect activity, stability, and immunogenicity and thus must be well-characterized, controlled, and monitored during development processes [20,21,81].…”
Biopharmaceuticals are highly complex molecules and also require high quality for safety and efficacy in human uses. For well-characterized products, the desired level of quality should be monitored and controlled during the manufacturing processes. A series of workflow for analytical characterization should be applied for product quality throughout those processes. In this chapter, several analytical techniques are introduced for assessing characteristics of biopharmaceuticals focusing on monoclonal antibodies (mAbs). Analytical characterization for primary structure was performed by mass spectrometry (MS), and assessment of post-translational modifications (PTMs) was done by conventional approaches. The analytical assessments were also done by multi-attribute method (MAM) approach using mass spectrometer (MS), and the performance of MAM was compared to conventional approaches. on the market results in gaining interest for drug development industry, and biopharmaceuticals are considered as fast growing and promising area for drug development [3][4][5][6].The approval of mAb-related products is dramatically increased in the recent years [6,7]. Over 90 mAb-related products are approved by European Medicines Agency (EMA) and US Food and Drug Administration (FDA). Those can be classified into mAb, Fc-fusion, Fab, antibody-drug conjugate (ADC), bispecific mAb (bsAb), and bispecific T cell engager (BiTE). Among them, mAbs are the major product, consisting of 77% of total. Others represent rest 23% of total, Fc-fusion (12%), ADC (5%), Fab (3%), bsAb (2%), and BiTE (1%), respectively. After the first approval of full-length mAb in 1998, mAbs are major product in the biopharmaceutical industry. This increasing number gives high revenue for pharmaceutical companies, and seven mAb-related products are positioned in top 10 drugs in the world, 2017, including Humira, Enbrel, Rituxan, Remicade, AVASTIN, Herceptin, and Lantus [8].Mylotarg is the first approved ADC in 2000, which combined a mAb targeting leukemic blast cells with a bacterial toxin (calicheamicin) [7,9]. ADC is a complex generated between a mAb and small molecule or a peptide, and mAb gives the selective delivery for targeting of cytotoxic drugs [1,[9][10][11]. Since the first approval, four additional ADC products are approved in Europe and USA. bsAb has two different antigen binding sites recognizing two different epitopes in a single mAb, and this dual specificity gives more specific targeting and higher efficacy [12][13][14]. Currently, three bsAbs are approved by EMA or US FDA. The first bsAb, Removab, was approved in 2009 but voluntarily withdrawn in 2013. Fc-fusion proteins are fusions of the IgG Fc domain with a desired linked protein, enhancing pharmacokinetic properties (serum half-life) and pharmacodynamics properties (ADCC and CDC) [6,15]. Following the first approval of Fc-fusion protein, Enbrel in 1998, eight Fc-fusion proteins are authorized for the marketing in the region of Europe and USA.Biosimilars, known as follow-on biologics, which follow t...
“…A second drawback of recombinant monoclonal antibodies is related to their production in cells of non-human origin (e.g., Chinese hamster ovary cells) or non-B-cell lineage (e.g., human embryonic kidney cells). The function of antibodies is strongly influenced by post-translational modifications (Li et al., 2015), which may differ between these cell lines and human B cells.…”
Summary
Vaccination approaches have generally focused on the antigen rather than the resultant antibodies generated, which differ greatly in quality and function between individuals. The ability to replace the variable regions of the native B cell receptor (BCR) heavy and light chain loci with defined recombined sequences of a preferred monoclonal antibody could enable curative adoptive cell transfer. We report CRISPR-mediated homologous recombination (HR) into the BCR of primary human B cells. Ribonucleoprotein delivery enabled editing at the model
CXCR4
locus, as demonstrated by T7E1 assay, flow cytometry, and TIDE analysis. Insertion via HR was confirmed by sequencing, cross-boundary PCR, and restriction digest. Optimized conditions were used to achieve HR at the BCR variable heavy and light chains. Insertion was confirmed at the DNA level, and transgene expression from the native BCR promoters was observed. Reprogramming the specificity of antibodies in the genomes of B cells could have clinical importance.
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