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- Brinson, Robert G., et al.
(författare)
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Enabling adoption of 2D-NMR for the higher order structure assessment of monoclonal antibody therapeutics
- 2019
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Ingår i: mAbs. - : Informa UK Limited. - 1942-0862 .- 1942-0870. ; 11:1, s. 94-105
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Tidskriftsartikel (refereegranskat)abstract
- The increased interest in using monoclonal antibodies (mAbs) as a platform for biopharmaceuticals has led to the need for new analytical techniques that can precisely assess physicochemical properties of these large and very complex drugs for the purpose of correctly identifying quality attributes (QA). One QA, higher order structure (HOS), is unique to biopharmaceuticals and essential for establishing consistency in biopharmaceutical manufacturing, detecting process-related variations from manufacturing changes and establishing comparability between biologic products. To address this measurement challenge, two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) methods were introduced that allow for the precise atomic-level comparison of the HOS between two proteins, including mAbs. Here, an inter-laboratory comparison involving 26 industrial, government and academic laboratories worldwide was performed as a benchmark using the NISTmAb, from the National Institute of Standards and Technology (NIST), to facilitate the translation of the 2D-NMR method into routine use for biopharmaceutical product development. Two-dimensional H-1,N-15 and H-1,C-13 NMR spectra were acquired with harmonized experimental protocols on the unlabeled Fab domain and a uniformly enriched-N-15, 20%-C-13-enriched system suitability sample derived from the NISTmAb. Chemometric analyses from over 400 spectral maps acquired on 39 different NMR spectrometers ranging from 500 MHz to 900 MHz demonstrate spectral fingerprints that are fit-for-purpose for the assessment of HOS. The 2D-NMR method is shown to provide the measurement reliability needed to move the technique from an emerging technology to a harmonized, routine measurement that can be generally applied with great confidence to high precision assessments of the HOS of mAb-based biotherapeutics.
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| 2. |
- Edgar, Rebecca J., et al.
(författare)
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Discovery of glycerol phosphate modification on streptococcal rhamnose polysaccharides
- 2019
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Ingår i: Nature Chemical Biology. - : Springer Science and Business Media LLC. - 1552-4450 .- 1552-4469. ; 15:5, s. 463-
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Tidskriftsartikel (refereegranskat)abstract
- Cell wall glycopolymers on the surface of Gram-positive bacteria are fundamental to bacterial physiology and infection biology. Here we identify gacH, a gene in the Streptococcus pyogenes group A carbohydrate (GAC) biosynthetic cluster, in two independent transposon library screens for its ability to confer resistance to zinc and susceptibility to the bactericidal enzyme human group IIA-secreted phospholipase A(2). Subsequent structural and phylogenetic analysis of the GacH extracellular domain revealed that GacH represents an alternative class of glycerol phosphate transferase. We detected the presence of glycerol phosphate in the GAC, as well as the serotype c carbohydrate from Streptococcus mutans, which depended on the presence of the respective gacH homologs. Finally, nuclear magnetic resonance analysis of GAC confirmed that glycerol phosphate is attached to approximately 25% of the GAC N-acetylglucosamine side-chains at the C6 hydroxyl group. This previously unrecognized structural modification impacts host-pathogen interaction and has implications for vaccine design.
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- Gray, Christopher J., et al.
(författare)
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Advancing Solutions to the Carbohydrate Sequencing Challenge
- 2019
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 141:37, s. 14463-14479
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Forskningsöversikt (refereegranskat)abstract
- Carbohydrates possess a variety of distinct features with stereochemistry playing a particularly important role in distinguishing their structure and function. Monosaccharide building blocks are defined by a high density of chiral centers. Additionally, the anomericity and regiochemistry of the glycosidic linkages carry important biological information. Any carbohydrate-sequencing method needs to be precise in determining all aspects of this stereodiversity. Recently, several advances have been made in developing fast and precise analytical techniques that have the potential to address the stereochemical complexity of carbohydrates. This perspective seeks to provide an overview of some of these emerging techniques, focusing on those that are based on NMR and MS-hybridized technologies including ion mobility spectrometry and IR spectroscopy.
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- Gulin, Sofia, et al.
(författare)
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Structural studies of S-7, another exocellular polysaccharide containing 2-deoxy-arabino-hexuronic acid
- 2001
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Ingår i: Carbohydrate Research. - 0008-6215 .- 1873-426X. ; 331:3, s. 285-290
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Tidskriftsartikel (refereegranskat)abstract
- The exocellular polysaccharide S-7, a heteropolysaccharide from Azotobacter indicus var, myxogenes has been studied using methylation analysis, Smith degradation, partial acid hydrolysis, NMR spectroscopy and mass spectrometry as the principal methods. It is concluded that the repeating unit has the following structure: -->4)-beta -D-Glcp-(1 -->4)-alpha -L-Rhap-(1 -->3)-beta -D-2-deoxy-arabino-HexpA-(1 --> 6 up arrow 1 beta -D-Glcp-(1 -->6)-beta -D-Glcp The absolute configuration of the deoxyhexuronic acid was deduced from H-1 NMR chemical shifts and is most likely D. Approximately two O-acetyl groups per repeating unit are present, one of which is presumably on the Rha residue. The structure bears great resemblance to another polysaccharide, recently studied, produced by Sphingomonas paucimobilis I-886.
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