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A rapid and quantitative technique for assessing IgG monomeric purity, calibrated with the NISTmAb reference material.

ABSTRACT: The fraction of intact monomer in a sample (moles/moles), the monomeric purity, is measured as a quality control in therapeutic monoclonal antibodies but is often unknown in research samples and remains a major source of variation in quantitative antibody-based techniques such as immunoassay development. Here, we describe a novel multiplex technique for estimating the monomeric purity and antigen affinity of research grade antibody samples. Light scattering was used to simultaneously observe the mass of antibody binding to biosensor surfaces functionalised with antigen (revealing Fab binding kinetics) or protein A/G (PAG). Initial estimates of monomeric purity in 7 antibody samples including a therapeutic infliximab biosimilar were estimated by observing a mass deficit on the PAG surface compared to the NISTmAb standard of high monomeric purity. Monomeric purity estimates were improved in a second step by observing the mass of antigen binding to the mass of antibody on the PAG surface. The NISTmAb and infliximab biosimilar displayed tightly controlled stoichiometries for antigen binding of 1.31?±?0.57 and 1.71?±?0.16 (95% confidence interval)-within the theoretical limit of 1-2 antigens per antibody depending on avidity. The other antibodies in the panel displayed antigen binding stoichiometries in the range 0.06-1.15, attributed to lower monomeric purity. The monomeric purity estimates were verified by electrospray ionization mass spectrometry (ESI), the gold standard technique for structural characterization of antibodies. ESI data indicated that the NISTmAb and infliximab biosimilar samples had monomeric purity values of 93.5% and 94.7%, respectively, whilst the research grade samples were significantly lower (54-89%). Our results demonstrate rapid quality control testing for monomeric purity of antibody samples (

SUBMITTER: Reader PP

PROVIDER: S-EPMC6718376 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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A rapid and quantitative technique for assessing IgG monomeric purity, calibrated with the NISTmAb reference material.

Reader Peter P PP Olkhov Rouslan V RV Reeksting Shaun S Lubben Anneke A Hyde Christopher J CJ Shaw Andrew M AM

Analytical and bioanalytical chemistry 20190802 24

The fraction of intact monomer in a sample (moles/moles), the monomeric purity, is measured as a quality control in therapeutic monoclonal antibodies but is often unknown in research samples and remains a major source of variation in quantitative antibody-based techniques such as immunoassay development. Here, we describe a novel multiplex technique for estimating the monomeric purity and antigen affinity of research grade antibody samples. Light scattering was used to simultaneously observe the ...[more]

PMID: 31375854

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Project description:RationaleThe stable carbon isotopic (δ13 C) reference material (RM) LSVEC Li2 CO3 has been found to be unsuitable for δ13 C standardization work because its δ13 C value increases with exposure to atmospheric CO2 . A new CaCO3 RM, USGS44, has been prepared to alleviate this situation.MethodsUSGS44 was prepared from 8 kg of Merck high-purity CaCO3 . Two sets of δ13 C values of USGS44 were determined. The first set of values was determined by online combustion, continuous-flow (CF) isotope-ratio mass spectrometry (IRMS) of NBS 19 CaCO3 (δ13 CVPDB = +1.95 milliurey (mUr) exactly, where mUr = 0.001 = 1‰), and LSVEC Li2 CO3 (δ13 CVPDB = -46.6 mUr exactly), and normalized to the two-anchor δ13 CVPDB-LSVEC isotope-delta scale. The second set of values was obtained by dual-inlet (DI)-IRMS of CO2 evolved by reaction of H3 PO4 with carbonates, corrected for cross contamination, and normalized to the single-anchor δ13 CVPDB scale.ResultsUSGS44 is stable and isotopically homogeneous to within 0.02 mUr in 100-μg amounts. It has a δ13 CVPDB-LSVEC value of -42.21 ± 0.05 mUr. Single-anchor δ13 CVPDB values of -42.08 ± 0.01 and -41.99 ± 0.02 mUr were determined by DI-IRMS with corrections for cross contamination.ConclusionsThe new high-purity, well-homogenized calcium carbonate isotopic reference material USGS44 is stable and has a δ13 CVPDB-LSVEC value of -42.21 ± 0.05 mUr for both EA/IRMS and DI-IRMS measurements. As a carbonate relatively depleted in 13 C, it is intended for daily use as a secondary isotopic reference material to normalize stable carbon isotope delta measurements to the δ13 CVPDB-LSVEC scale. It is useful in quantifying drift with time, determining mass-dependent isotopic fractionation (linearity correction), and adjusting isotope-ratio-scale contraction. Due to its fine grain size (smaller than 63 μm), it is not suitable as a δ18 O reference material. A δ13 CVPDB-LSVEC value of -29.99 ± 0.05 mUr was determined for NBS 22 oil.

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A rapid and quantitative technique for assessing IgG monomeric purity, calibrated with the NISTmAb reference material.

Publications

A rapid and quantitative technique for assessing IgG monomeric purity, calibrated with the NISTmAb reference material.

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