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Applications of NMR for assessing the higher order structure of protein
therapeuticsDavid Keire
Laboratory Chief (Acting) Branch 1United States Food and Drug Administration
CDER/OPQ/OTR/DPA12/05/2016
This presentation reflects the views of the author and should not be construed to represent FDA’s views or policies
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NMR and the “state” of the protein
Aubin et al., Chap 13 in Biophysical Characterization of Proteins in Developing Biopharmaceuticals, Berkowitz and Houde eds., Pages 341-383, 2015
HGH vs. G-CSFSimilar size but different linewidths
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HGH• Self-associated at sub-millimolar concentrations
• Self-association increased with concentration, was reversible and exhibits fast exchange on the NMR timescale.
• Only weak, non-covalent interactions were observed with soluble dimer, trimer and tetramer aggregates present.
• No larger insoluble aggregates were observed.
Jensen et al., Proteins, Vol 73(1), 161-172, 2008
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Correlation time and relaxation
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Things that give you fat lines
• Slow rotational tumbling
• Solution viscosity
• Chemical Exchange between states– Intra-molecular (e.g., cis-trans isomerism)
– Inter-molecular (e.g., with bulk solvent and/or with aggregated forms)
• Paramagnetic ions
• Non-homogenous magnetic fields
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6http://www.bioc.aecom.yu.edu/labs/girvlab/nmr/course/COURSE_2012/Lecture_ExchangeDynamics_2012.pdf
Where exchange occurs between species of unequal population the resulting spectra reflect the population weighted average of those states.
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Chemical Exchange for Aggregates• An “NMR invisible signal” (e.g., a larger aggregated
protein form) can impact the measurement of an NMR visible signal (monomer form).
• Probe by dark exchange saturation transfer (DEST) or paramagnetic relaxation experiments (PRE).
Work coming from the Clore group at NIH e.g., Anthis and Clore, Q. Rev. Biophys., 48(1), 35-116, 2015Review for beta-amyloid studies Karamanos et al., Prog NMR Spectr., Vol 88-89, 86-104, 2015
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Protein Timescales
10-15 10-12 10-9 10-6 10-3 1 103
Bond Vibration
Side Chain Rotation
Molecular Tumbling
Domain Motion
Folding
Residual Dipolar Coupling (RDC)
Paramagnetic Relaxation Enhancement (PRE)
Room Temperature
NMR
CPMG
DOSY
Time (s)
HDX
NMR Land
Association/Aggregation
Lineshape
DEST
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Rituximab vs Infliximab
Chen et al., JPBA, 128, 398-407, 2016
83% Identity91% Similarity
Fab RMSDsBB 1.6 ÅSC 2.2 Å
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1D Proton NMR at 600 MHz
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Reproducibility
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Size matters
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PCA
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Insulin Structure and Equilibrium
Journal of Molecular Biology [2002, 318(2):479-490]
α - chain
β - chain
Zn++Phenol
Pdb:1mso,
1ev6, 1lph
Spectra peak position: sequence, 2º and 3º structure.
Peak line-width and intensity: 4º structure, concentration,
equilibrium and kinetics.
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Rapid Insulins in US market
Drug
Name
Pharma.
Company
API
Conc. (0.6 mM)
M.W.
(Da)
Organic
Excipients
Conc.
(mM)
Novolin RNovo
Nordisk
Insulin
Regular5808
glycerol
metacresol
173.7
27.74
Humulin R LillyInsulin
Regular5808
glycerin
metacresol
173.7
23.12
ApidraSanofi-
Aventis
Glulisine
B3: Asn->Lys
B29: Lys->Glu
5823
Metacresol tromethamine
polysorbate-20
29.13
49.52
8.15
NovologNovo
Nordisk
Aspart
B28: Pro->Asp5826
glycerol
Phenol
metacresol
173.7
15.94
15.91
Humalog Lilly
Lispro
B28: Pro->Lys;
B29: Lys->Pro.
5808glycerin
metacresol
173.7
29.1315
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1D 1H spectra on insulin drug product
HumulinR ® (black)
NovolinR ® (red)
95% Insulin drug
formulations + 5% D2O
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2D DOSY Spectra
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Novolin R (Red)Humulin R (Blue)
insulin
excipient
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Diffusion coefficient for insulin
www.fda.gov
3 lots for each product, dashed line are computer simulation results of diffusion coefficients
Inter-lot STD
1.8% 5.5% 5.2% 0.13% 1.9%
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Summary
• 2D DOSY separates API signal from excipient and provides data for protein quaternary and quinary structure data.
• The observed insulin NMR signal represents exchange average between dimer and hexamer. Monomer does not exist in formulation.
• The DOSY method was highly reproducible and demonstrated 0.13% variation in repeated measurements D0 for Novolin R products.
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2D 1H-13C HSQC on insulin drug product
95% Insulin drug
formulations + 5% D2O
m-cresol
m-cresol
glycerol
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2D 1H-13C
MS-HSQC on Methyl region
NovolinR ®
Exp. time = 12 Hr
Xiaoquing Chang et.al., Biochemistry, 1997, 36(31), 9409-9422.Kang Chen et al., JMR, 251, 65-70, 2015
BMRB
Chen, Freedberg, Keire. JMR, 2015 (251) 65-70.
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Overlay of
methyl MS-
HSQC
NovolinR®HumulinR®
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PCA score plot on 1D/2D spectra
Inter-brand
Intra-brand
2D PCA method: Ghasriani et al. NBT Vol 34(2) Feb 2016
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24www.fda.gov
Conclusion
• 1D NMR can inform upon the state of a protein in solution including self-association and aggregation. Excipients can sometimes interfere Calibration is needed for quantification.
• 2D NMR spectra, both HSQC and DOSY, can be applied to drug formulations directly.
• PCA is a valuable tool to identify differences between the complex spectra of comparators.
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Acknowledgements:Kang Chen
Sharadrao PatilHouman Ghasriani
Diana LongMichael Karfunkle
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