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Current Pharmaceutical Analysis


ISSN (Print): 1573-4129
ISSN (Online): 1875-676X

Research Article

Protein Species of Origin Determination By NMR Relaxometry

Author(s): Gregory K. Webster*

Volume 19, Issue 9, 2023

Published on: 07 November, 2023

Page: [687 - 694] Pages: 8

DOI: 10.2174/0115734129246067230921050607

Price: $65


Aims: The aim of this project was to develop a QC friendly and efficient method of protein species of origin identification to replace more costly mass spectrometric based methods currently being used for this testing.

Background: NMR relaxation measurements with proteins in aqueous solutions exploit the fast chemical exchange between water and exposed NH and OH protons of amino acid side chains in the folded protein structure unique to each biologic drug. Implementation of this technique has led to routine testing for authentication and forensics of biopharmaceuticals, determination of moisture content in lyophilized protein formulations and aggregation of proteins in solution. For small molecule applications, TD-NMR can detect if solvents are received neat or tainted with moisture, impurities, or denaturants.

Objective: The objective of this study was to evaluate the ability of NMR Relaxation measurements to differentiate between sources of Albumin proteins as a rapid QC test. Evaluation of differences in molecular mobility between components in a solution as reflected in the longitudinal (T1) and transverse (T2) relaxation times of protons demonstrate that NMR relaxation techniques can distinguish between different albumin sources of origin.

Methods: Representative albumin proteins from differing sources of origin were studied. Using bovine serum albumin (BSA) as the target species of origin, NMR relaxation techniques as well as chemometric modeling were used to evaluate the use of this technique for protein source of origin identification.

Results: NMR Relaxation using benchtop instrumentation showed that the bovine albumin species of origin can be distinguished from porcine, chicken egg white and sheep sources of origin. Goat albumin selectivity remained questionable and BSA cannot be distinguished from human or rabbit sources of origin within the representative variability.

T2 transverse relaxation results were significantly more discriminating for protein source identification than the T1 longitudinal relaxation result by itself. The T1 longitudinal relaxation result did not contribute significantly to this investigation. However, fusing the T1 data with the T2 transverse relaxation results and using larger data sets merit further evaluation in the hope of achieving additional selectivity.

Conclusion: While additional lots are needed for more definitive results, this preliminary evaluation of using NMR Relaxation demonstrated the capability for the source of origin species discrimination and identification using benchtop NMR instrumentation.

Keywords: NMR relaxation, relaxometry, TD-NMR, protein source of origin, albumin identification, transverse relaxation.

Graphical Abstract
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