Analytical NMR approaches
for biopharmaceutical product characterisation
Mark Howard
14 April 2015
AIMS
Has NMR spectroscopy been overlooked in biopharmaceutical analysis?
- As an early warning detection device for product issues
- As a general analytical resource to monitor product integrity
What should we monitor?
Our key aim is to detect soluble aggregates using NMR spectroscopy
In biopharma, aggregation is a development and regulatory ‘headache’
What should we monitor?
We could also use NMR to monitor both integrity and fidelity of products
- there are many processes that can influence the product
What should we monitor?
Process monitoring is also possible by NMR
- product fidelity
- metabolic profile of feedstock
Why use NMR?
NMR is a subatomic analytical spectroscopy
- sounds very cool
NMR ‘tunes’ to different nuclear isotopes
- e.g. 1H, 13C, 15N, 19F, 31P
The ‘response’ from NMR is very sensitive:
- chemical structure
- protein environment (fold)
- size of species under investigation
- can be quantitative
- capable of registering very subtle changes
NMR and molecular size: is it an Achille’s Heel?
Direct detection of a protein
Increasing folded protein molecular weight
NMR peak gets broader  harder to detect
Indirect detection of a protein using a ‘small’ reporter molecule (using STD)
Increasing folded protein molecular weight
NMR peak gets taller  easy to detect and quantifiable
Saturation Transfer Difference (STD) NMR
Receptor-specific proton saturation
1
Receptor
koff
kon
Saturation transfer efficiency  as Mwt 
1 3
2 Ligand
4
5 6
6
H STDdiff spectrum
2
3
1
4
5
 method for detecting soluble aggregates?
CAVEAT: koff and kon must be comparable from monomer to multimer
WORK AROUND: use a non-specific water soluble ligand probe with high μM Kd
Glucose Oxidase (Aspergillus Niger) 65.7 kDa monomer
Non-reducing SDS PAGE – 1 h @ 50°C
pH
4
5
6
7
8
9
10
11
12
250
Dimer
150
100
GOX
75
4
37
3
STDamp
50
Native PAGE– 1 h @ 50°C
4
5
6
7
8
9
10
11
12
2
1
0
2
 Soluble
aggregates
5
4
6
8
10
12
CONTROL (no incubation)
4
 GOX
STDamp
pH
STD AMP FACTOR (50oC incubation)
5
3
2
1
0
2
4
6
8
Incubation pH
10
12
Gox Aggregates
Gel filtration of GOX after incubation at 50°C for 1 h
Native GOX
̴170 kDa
190
pH 4
pH 5
pH 6
pH 7
pH 8
pH 9
pH 10
pH 11
pH 12
A280 (mAu)
140
90
Aggregates
600 – 1400 kDa
Degradation
products
̴ 4000 Da
40
-10
0
50
100
150
Elution volume (mL)
200
250
300
Glucose Oxidase (Aspergillus Niger) 65.7 kDa
STD signal from the probe molecule  as aggn 
STD signal changes when the protein denatures
pH 12
Probe molecule
pH 10
pH 8
50 μM Gox, 2 mM probe
pH 6
120 minute experiment @ 25°C
pH 4
Works with different probes
IgG (Human): 150-170 kDa
1 hr incubation @ 60oC
pH
4
5
6
7
8
9
10
11
12 CTRL
Aggregates 
GEL Filtration eg. pH 9
25.000
Heavy chain
50kDa
20.000
538 kDa
15.000
UV (mAU)
Light chain
23kDa
227 kDa
1358 kDa
10.000
GF confirms higher order aggregates as pH 
denaturation at pH 12
2256 kDa
5.000
CTRL is a pH 7 sample without incubation
Low concentration of aggregates
0.000
0.00
50.00
100.00
150.00
200.00
Elution Volume (ml)
250.00
300.00
IgG (Human): STD NMR DATA
14
Modest  as pH  (60C incubation)
12
Reflects small aggregate population
STD amp signal
10
Uses a different probe molecule to GOx
8
6
20 μM IgG, 2 mM probe: 120 min expt
4
Currently being optimised
2
Is 60°C incubation enough?
0
4
5
6
7
8
9
pH
Vermeer and Willem Norde, Biophys J. 78, 394-404, (2000)
DIRECT PROTEIN OBSERVE: This is what happens to 1H NMR as Mwt 
13 kDa
24 kDa
40 kDa
52 kDa
Practical example of line broadening with molecular size
IgG (Human): FOLDING & INTEGRITY FROM 1D 1H NMR
H2O
110 μM IgG (Sigma) in 20 mM PBS (50 mM salt) at 25°C
7 minute acquisition
Destabilising IgG
Liu and May, mAbs, 4, 17-23, (2012)
Many S-S bonds: can be destabilised by adding reductant (e.g. dithiothreitol-DTT)
IgG (Human): ± DTT
Can you see the differences?
+ 20 mM DTT
CONTROL
IgG (Human): ± DTT (zoom in)
N
H
N
H
Aro
N
H
+ 20 mM DTT
CONTROL
CH3
IgG (Human): Thermal denaturation
70°C
65°C
55°C
45°C
35°C
25°C
Differences are there but hard to see
H2O  as T
IgG (Human): Thermal denaturation with NMR editing
1H
NMR motion edited: shows increased molecular motion as T
BUT also highlights denaturation
70°C
65°C
55°C
45°C
35°C
25°C
CONCLUSIONS
Has NMR spectroscopy been overlooked in biopharmaceutical analysis?
- As an early warning detection device for product issues
- NMR shows promise in the early detection of soluble aggregates
- As a general analytical resource to monitor product integrity
- Minor and major changes can be observed in IgG samples
Thanks…
KENT
Robyn Tucker
Denisa Doko
Michelle Rowe
Mark Smales
Richard Williamson
Gary Robinson
LGC
John Warren
Helen Parkes
FUNDING
EPSRC
BBSRC Ideas factory
Wellcome Trust