Abstract:The successful development and regulatory approval of originator and biosimilar therapeutic proteins requires a systems approach to upstream and downstream processing as well as product characterization and quality control. Innovation in process design and control, product characterization strategies, and data integration represent an ecosystem whose concerted advancement may reduce time-to-market and further improve comparability and biosimilarity programs. The biopharmaceutical community has made great strid… Show more
“…Here we tested whether FUT8 can tolerate the three fluorescent fucoses and detect its substrate glycans on Cantuzumab that was prepared from FUT8 knockout cell line and the reference monoclonal antibody from the National Institute of Standards and Technology (NIST mAb, material 8671). NIST mAb is a humanized IgG1κ monoclonal antibody 22 . IgG antibodies are known to contain an N-glycan site on their heavy chains 23 .…”
Like sialylation, fucose usually locates at the non-reducing ends of various glycans on glycoproteins and constitutes important glycan epitopes. Detecting the substrate glycans of fucosyltransferases is important for understanding how these glycan epitopes are regulated in response to different growth conditions and external stimuli. Here we report the detection of these glycans via enzymatic incorporation of fluorescent tagged fucose using fucosyltransferases including FUT2, FUT6, FUT7, and FUT8 and FUT9. More specifically, we describe the detection of substrate glycans of FUT8 and FUT9 on therapeutic antibodies and the detection of high mannose glycans on glycoproteins by enzymatic conversion of high mannose glycans to the substrate glycans of FUT8. By establishing a series of precursor glycans, we also demonstrate the substrate specificities of FUT8. Furthermore, using simultaneous enzymatic incorporation of both fluorescent sialic acids and fluorescent fucoses, we demonstrate the interplay between fucosylation and sialylation.
“…Here we tested whether FUT8 can tolerate the three fluorescent fucoses and detect its substrate glycans on Cantuzumab that was prepared from FUT8 knockout cell line and the reference monoclonal antibody from the National Institute of Standards and Technology (NIST mAb, material 8671). NIST mAb is a humanized IgG1κ monoclonal antibody 22 . IgG antibodies are known to contain an N-glycan site on their heavy chains 23 .…”
Like sialylation, fucose usually locates at the non-reducing ends of various glycans on glycoproteins and constitutes important glycan epitopes. Detecting the substrate glycans of fucosyltransferases is important for understanding how these glycan epitopes are regulated in response to different growth conditions and external stimuli. Here we report the detection of these glycans via enzymatic incorporation of fluorescent tagged fucose using fucosyltransferases including FUT2, FUT6, FUT7, and FUT8 and FUT9. More specifically, we describe the detection of substrate glycans of FUT8 and FUT9 on therapeutic antibodies and the detection of high mannose glycans on glycoproteins by enzymatic conversion of high mannose glycans to the substrate glycans of FUT8. By establishing a series of precursor glycans, we also demonstrate the substrate specificities of FUT8. Furthermore, using simultaneous enzymatic incorporation of both fluorescent sialic acids and fluorescent fucoses, we demonstrate the interplay between fucosylation and sialylation.
“…Some of these additional bands are likely due to alternative or incomplete disulfide bond formation, as was shown for the mammalian-expressed NISTmAb. 26 10.1080/19420862.2018.1496879-F0005Figure 5.The eNISTmAb forms a tetrameric complex.A) The eNISTmAb protein solution (300 μg) and molecular mass standards were subjected to Superdex-200 gel filtration analysis. The protein concentration in each eluted fraction was measured by ultraviolet absorption at 280nm.…”
The widespread use of monoclonal antibodies (mAbs) as a platform for therapeutic drug development in the pharmaceutical industry has led to an increased interest in robust experimental approaches for assessment of mAb structure, stability and dynamics. The ability to enrich proteins with stable isotopes is a prerequisite for the in-depth application of many structural and biophysical methods, including nuclear magnetic resonance (NMR), small angle neutron scattering, neutron reflectometry, and quantitative mass spectrometry. While mAbs can typically be produced with very high yields using mammalian cell expression, stable isotope labeling using cell culture is expensive and often impractical. The most common and cost-efficient approach to label proteins is to express proteins in Escherichia coli grown in minimal media; however, such methods for mAbs have not been reported to date. Here we present, for the first time, the expression and purification of a stable isotope labeled mAb from a genetically engineered E. coli strain capable of forming disulfide bonds in its cytoplasm. It is shown using two-dimensional NMR spectral fingerprinting that the unlabeled mAb and the mAb singly or triply labeled with 13C, 15N, 2H are well folded, with only minor structural differences relative to the mammalian cell-produced mAb that are attributed to the lack of glycosylation in the Fc domain. This advancement of an E. coli-based mAb expression platform will facilitate the production of mAbs for in-depth structural characterization, including the high resolution investigation of mechanisms of action.
“…Our motivation to develop a model IA-MS assay was justified by its future use to map epitopes of monoclonal antibodies and characterize the endogenous polyclonal antibodies. Interaction of NISTmAb with preF protein was chosen as an excellent model due to: (i) high affinity of binding (Kd = 4 nM); (ii) a known epitope NSELLSLINDMPITNDQKKLMSNN 59 ; (iii) high affinity of binding to the linear peptide epitope (66 nM) 60 ; (iv) availability and affordability of a high-quality purified NISTmAb standard; and (v) availability of several monoclonal antibodies with different affinities (palivizumab, motavizumab, and nirsevimab 61 ) which could be used as a model to separate antibody mixtures by their affinities, as we previously demonstrated for combinatorial DNA libraries [62][63][64][65][66][67][68][69][70][71] . We demonstrated that NISTmAb 8671 was efficiently enriched with preF coated onto microplates (Fig.…”
Section: Development Of Ia-ms Assay To Measure the Interaction Of Nis...mentioning
SUMMARYRecent advances in proteomics and mass spectrometry facilitated the in-depth characterization of monoclonal antibodies and enabled innovative approaches for the quantification of polyclonal antibodies generated against numerous antigens. Human respiratory syncytial virus (RSV) is a contagious respiratory pathogen often manifested as a common cold infection in adults and more serious symptoms in infants and the elderly population. Here, we used a reference IgG1κ monoclonal antibody NISTmAb 8671 and its affinity interaction with an RSV fusion glycoprotein F as a model to develop the proteomic toolbox for identification, quantification, and characterization of polyclonal antibodies. Our toolbox integrated a variety of proteomic and mass spectrometry approaches for accurate mass measurements of antibody fragments, antibody digestion with the complimentary proteases (trypsin, asparaginase, and proalanase), immunoaffinity enrichments, and bottom-up or middle-down proteomics. We measured absolute concentrations of anti-RSV antibody isotypes and subclasses in 69 serum samples of healthy individuals and revealed IgG1 (2,580 ng/mL), IgA1 (280 ng/mL), and IgM (180 ng/mL) as the most abundant isotypes. Interestingly, we also identified the presence of IgG2 (74 ng/ml), IgG4 (4.9 ng/mL) and IgA2 (5.5 ng/mL) antibodies. Interactome measurements detected the consistent co-precipitation of C1q complement complexes. Repertoire profiling of the variable regions of polyclonal antibodies revealed the frequent use of IGHV3 subgroup genes, while IGHV5-51 was the most abundant single gene of the anti-RSV polyclonal antibody response. The presented toolbox will facilitate the in-depth characterization of polyclonal antibodies and pave the way to quantitative approaches in serological studies and precision immunology.
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