Expression and characterization of 15N-labeled human C-reactive protein in Escherichia coli and Pichia pastoris for use in isotope-dilution mass spectrometry
“…1 our culturing methods successfully yielded 15 N labelled vegetative cells and 14 N spores. For a number of identified 15 N labelled vegetative cell peptides, the 15 N label content has been calculated based on their mass spectrometric isotope patterns using the NIST isotope calculator (32). This shows that the present metabolic labelling method achieves a 15 N label content of ≥ of 99.5%, which is amply sufficient for accurate protein quantification.…”
“…1 our culturing methods successfully yielded 15 N labelled vegetative cells and 14 N spores. For a number of identified 15 N labelled vegetative cell peptides, the 15 N label content has been calculated based on their mass spectrometric isotope patterns using the NIST isotope calculator (32). This shows that the present metabolic labelling method achieves a 15 N label content of ≥ of 99.5%, which is amply sufficient for accurate protein quantification.…”
“…The same protein load (based on A 280 ) was analyzed for all four samples, and the lower apparent signal to noise is thought to be a result of increased heterogeneity in the non-originator samples, as will be discussed in more detail below. Peak identifications were made by comparing the measured zero-charge state mass to theoretical values based on purported amino acid/glycan compositions and calculated using the NIST Mass and Fragment calculator (Table 1 25 ) (Software available at https://www.nist.gov/services-resources/software/nist-mass-and-fragment-calculator-software). 10.1080/19420862.2018.1486355-F0005Figure 5.Deconvoluted intact mass spectra of non-originator NISTmAbs and RM 8671.Peaks marked with a * indicated the presence of a C-terminal lysine on the previous glycoform.…”
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 strides to this end, yet there currently exists no pre-competitive monoclonal antibody (mAb) expression platform for open innovation. Here, we describe the development and initial expression of an intended copy of the NISTmAb using three non-originator murine cell lines. It was found that, without optimization and in culture flasks, all three cell lines produce approximately 100 mg mAb per liter of culture. Sodium dodecyl sulfate polyacrylamide gel electrophoresis, size-exclusion chromatography, nuclear magnetic resonance spectroscopy, intact mass spectrometry, and surface plasmon resonance were used to demonstrate that the products of all three cell lines embody quality attributes with a sufficient degree of sameness to the NISTmAb Reference Material 8671 to warrant further bioreactor studies, process improvements and optimization. The implications of the work with regard to pre-competitive innovation to support process design and feedback control, comparability and biosimilarity assessments, and process analytical technologies are discussed.
“…However, NISTmAb has a pyroglutamate (pGlu) at the N-terminus of the heavy chain, which is a frequently observed cyclization of the gene-encoded N-terminal glutamine [18–20]. Accounting for the pyroglutamate (−18 Da) and five disulfides (four intrachain and one between chains, thus, losing ten hydrogens per Fab (−10 Da) [7,21,22]. Hence, the observed and predicted mass of the Fab fragment are in accord and confirm that papain cleaves NISTmAb after CDKTH 227 .…”
Monoclonal antibody pharmaceuticals are the fastest-growing class of therapeutics, with a wide range of clinical applications. To assure their safety, these protein drugs must demonstrate highly consistent purity and stability. Key to these objectives is higher order structure measurements validated by calibration to reference materials. We describe preparation, characterization, and crystal structure of the Fab fragment prepared from the NIST Reference Antibody RM 8671 (NISTmAb). NISTmAb is a humanized IgG1κ antibody, produced in murine cell culture and purified by standard biopharmaceutical production methods, developed at the National Institute of Standards and Technology (NIST) to serve as a reference material. The Fab fragment was derived from NISTmAb through papain cleavage followed by protein A based purification. The purified Fab fragment was characterized by SDS-PAGE, capillary gel electrophoresis, multi-angle light scattering, size exclusion chromatography, mass spectrometry, and x-ray crystallography. The crystal structure at 0.2 nm resolution includes four independent Fab molecules with complete light chains and heavy chains through Cys 223, enabling assessment of conformational variability and providing a well-characterized reference structure for research and engineering applications. This nonproprietary, publically available reference material of known higher-order structure can support metrology in biopharmaceutical applications, and it is a suitable platform for validation of molecular modeling studies.
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