Abstract
Protein therapeutics have numerous critical quality attributes (CQA) that must be evaluated to ensure safety and efficacy, including the requirement to adopt and retain the correct three-dimensional fold without forming unintended aggregates. Therefore, the ability to monitor protein higher order structure (HOS) can be valuable throughout the lifecycle of a protein therapeutic, from development to manufacture. 2D NMR has been introduced as a robust and precise tool to assess the HOS of a protein biotherapeutic. A common use case is to decide whether two groups of spectra are substantially different, as an indicator of difference in HOS. We demonstrate a quantitative use of principal component analysis (PCA) scores to perform this decision-making, and demonstrate the effect of acquisition and processing details on class separation using samples of NISTmAb monoclonal antibody Reference Material subjected to two different oxidative stress protocols. The work introduces an approach to computing similarity from PCA scores based upon the technique of histogram intersection, a method originally developed for retrieval of images from large databases. Results show that class separation can be robust with respect to random noise, reconstruction method, and analysis region selection. By contrast, details such as baseline distortion can have a pronounced effect, and so must be controlled carefully. Since the classification approach can be performed without the need to identify peaks, results suggest that it is possible to use even more efficient measurement strategies that do not produce spectra that can be analyzed visually, but nevertheless allow useful decision-making that is objective and automated.
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Availability of data and materials
Example processing and analysis scripts, and the spectral data used in the work will be posted at the NISTmAb NMR home page: https://www.ibbr.umd.edu/groups/nistmab-nmr. The NISTmAb Reference Material is available for purchase from NIST: https://www-s.nist.gov/srmors/view_detail.cfm?srm=8671.
Software availability
The work makes use of the following software, which is all also available on the NMRbox cloud computing platform: NMRbox: https://www.nmrbox.org. NMRPipe: https://www.ibbr.umd.edu/nmrpipe/install.html. SMILE: https://spin.niddk.nih.gov/bax/software/smile. hmsIST: http://gwagner.med.harvard.edu/intranet/hmsIST (download by request). NESTA: http://nestanmr.com (download by request).
Abbreviations
- 1D:
-
One-dimensional
- 2D:
-
Two-dimensional
- CQA:
-
Critical quality attribute
- DSS:
-
3-(Trimethylsilyl) propane-1-sulfonate
- gHSQC:
-
Gradient-selected heteronuclear single quantum coherence spectrum
- HOS:
-
Higher order structure
- mAb:
-
Monoclonal antibody
- NMR:
-
Nuclear magnetic resonance spectroscopy
- NUS:
-
Non-uniformly sampled
- PCA:
-
Principal component analysis
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Brinson, R.G., Elliott, K.W., Arbogast, L.W. et al. Principal component analysis for automated classification of 2D spectra and interferograms of protein therapeutics: influence of noise, reconstruction details, and data preparation. J Biomol NMR 74, 643–656 (2020). https://doi.org/10.1007/s10858-020-00332-y
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DOI: https://doi.org/10.1007/s10858-020-00332-y