Assessment of the Three-Dimensional Structure of Recombinant Protein Therapeutics by NMR Fingerprinting:  Demonstration on Recombinant Human Granulocyte Macrophage-Colony Stimulation Factor

Aubin, Yves; Gingras, Geneviève; Sauvé, Simon

doi:10.1021/ac7026222

Cited by 62 publications

(46 citation statements)

References 12 publications

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“…The spectral assignment and the analysis were performed using the Sparky 3.113 software48. The spectral assignments for the filgrastim samples were performed using the previously available chemical shift assignment databases using both the chemical shift and pattern matching121549505152.…”

Section: Methodsmentioning

confidence: 99%

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Biosimilar structural comparability assessment by NMR: from small proteins to monoclonal antibodies

Japelj¹,

Ilc

Marušič³

et al. 2016

Sci Rep

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Biosimilar drug products must have a demonstrated similarity with respect to the reference product’s molecules in order to ensure both the effectiveness of the drug and the patients’ safety. In this paper the fusion framework of a highly sensitive NMR fingerprinting approach for conformational changes and mathematically-based biosimilarity metrics is introduced. The final goal is to translate the complex spectral information into biosimilarity scores, which are then used to estimate the degree of similarity between the biosimilar and the reference product. The proposed method was successfully applied to a small protein, i.e., filgrastim (neutropenia treatment), which is the first biosimilar approved in the United States, and a relatively large protein, i.e., monoclonal antibody rituximab (lymphoma treatment). This innovative approach introduces a new level of sensitivity to structural changes that are induced by, e.g., a small pH shift or other changes in the protein formulation.

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Section: Methodsmentioning

confidence: 99%

“…A limited number of studies were so far published where authors used NMR fingerprint spectra to study higher order protein structure (HOS) and compare it to the reference product910111213. Aubin Y. et al .…”

mentioning

confidence: 99%

Biosimilar structural comparability assessment by NMR: from small proteins to monoclonal antibodies

Japelj¹,

Ilc

Marušič³

et al. 2016

Sci Rep

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“…These can bring challenges to the application of spectroscopic methods, in particular NMR spectroscopy, to assess the conformation of the drug substance. The relatively low sensitivity of NMR often requires the use of more than one dose in order to obtain a sufficient concentration of the protein (6). The sample can either be prepared from the isolation of the drug substance, or from the direct concentration an appropriate number of vials to obtain the desired amount.…”

Section: Titration Of Polysorbate-80mentioning

confidence: 99%

Monitoring Effects of Excipients, Formulation Parameters and Mutations on the High Order Structure of Filgrastim by NMR

et al. 2015

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The effects of pH variation on the amide chemical shifts suggest the presence of cation-pi interactions between His-79 and Trp-118, and His-156-Trp-58-His-52 that stabilizes the conformation at low pH. This may be associated with a small local conformational change. The NMR data showed that polysorbate does not interact significantly with filgrastim thus allowing the collection of spectra in the presence of 20 times the formulation concentration in the sample. However, at higher detergent concentrations a reduction of signal intensity is observed. Conclusions The NMR fingerprint assay applied to filgrastim (Neupogen® and a CRS from the European Pharmacopeia (EP)) provided residue specific information of the structure of the drug substance. In addition to current methods, the ability to assess the conformation with a high degree of resolution can greatly facilitate comparability exercises.

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“…[1][2][3][4][5][6][7][8] Two-dimensional hetero-nuclear 1 H-X (X = 15 N or 13 C) spectral patterns are sensitive to chemical structure and therefore can detect structural change at the level of individual nuclei. These methods are superior to homo-nuclear two-dimensional spectra because of their increased chemical shift dispersion, less homo-nuclear J-splitting, and concomitant reduced peak overlap.…”

Section: Introductionmentioning

confidence: 99%

“…These methods are superior to homo-nuclear two-dimensional spectra because of their increased chemical shift dispersion, less homo-nuclear J-splitting, and concomitant reduced peak overlap. [2] Twodimensional methods have become even more powerful with the advent of multiplicityedited 1 H-13 C HSQC, HMQC, HMBC and H2BC experiments. [9][10][11][12][13][14][15][16][17][18][19] Multiplicity-edited pulse sequences use a dedicated period of 1/ 1 J CH to edit CH 2 and CH/CH 3 peaks into the opposite phases in the same spectrum.…”

Section: Introductionmentioning

confidence: 99%

NMR profiling of biomolecules at natural abundance using 2D 1H–15N and 1H–13C multiplicity-separated (MS) HSQC spectra

Kang

Freedberg

Keire

2015

Journal of Magnetic Resonance

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2D NMR (1)H-X (X=(15)N or (13)C) HSQC spectra contain cross-peaks for all XHn moieties. Multiplicity-edited(1)H-(13)C HSQC pulse sequences generate opposite signs between peaks of CH(2) and CH/CH(3) at a cost of lower signal-to-noise due to the (13)C T(2) relaxation during an additional 1/(1)JCH period. Such CHn-editing experiments are useful in assignment of chemical shifts and have been successfully applied to small molecules and small proteins (e.g. ubiquitin) dissolved in deuterated solvents where, generally, peak overlap is minimal. By contrast, for larger biomolecules, peak overlap in 2D HSQC spectra is unavoidable and peaks with opposite phases cancel each other out in the edited spectra. However, there is an increasing need for using NMR to profile biomolecules at natural abundance dissolved in water (e.g., protein therapeutics) where NMR experiments beyond 2D are impractical. Therefore, the existing 2D multiplicity-edited HSQC methods must be improved to acquire data on nuclei other than (13)C (i.e.(15)N), to resolve more peaks, to reduce T(2) losses and to accommodate water suppression approaches. To meet these needs, a multiplicity-separated(1)H-X HSQC (MS-HSQC) experiment was developed and tested on 500 and 700 MHz NMR spectrometers equipped with room temperature probes using RNase A (14 kDa) and retroviral capsid (26 kDa) proteins dissolved in 95% H(2)O/5% D(2)O. In this pulse sequence, the 1/(1)JXH editing-period is incorporated in to the semi-constant time (semi-CT) X resonance chemical shift evolution period, which increases sensitivity, and importantly, the sum and the difference of the interleaved (1)J(XH)-active and the (1)J(XH)-inactive HSQC experiments yield two separate spectra for XH(2) and XH/XH(3). Furthermore we demonstrate improved water suppression using triple xyz-gradients instead of the more widely used z-gradient only water-suppression approach.

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Assessment of the Three-Dimensional Structure of Recombinant Protein Therapeutics by NMR Fingerprinting: Demonstration on Recombinant Human Granulocyte Macrophage-Colony Stimulation Factor

Cited by 62 publications

References 12 publications

Biosimilar structural comparability assessment by NMR: from small proteins to monoclonal antibodies

Biosimilar structural comparability assessment by NMR: from small proteins to monoclonal antibodies

Monitoring Effects of Excipients, Formulation Parameters and Mutations on the High Order Structure of Filgrastim by NMR

NMR profiling of biomolecules at natural abundance using 2D 1H–15N and 1H–13C multiplicity-separated (MS) HSQC spectra

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