2D NMR can provide precise structural fingerprints of biologics like monoclonal antibodies. NMR allows assignment of signals to specific regions of these complex molecules. While challenges include sensitivity and spectral complexity, selective pulse sequences and signal subtraction techniques can remove interfering excipient signals with minimal loss of sensitivity. Glycan remodeling studies examined how 2D NMR fingerprints change with modifications like removing or trimming glycans, correlated to the native structure. Multivariate analysis methods like PCA can automate comparison of modified structures to the native fingerprint without manual spectral analysis.

1. Quality assessment of biologics:
higher order structure analysis
using 2D NMR
LAKSHYA RAJ KHATRI

2. What is a biologic drug (biologics)?
• A biologic drug (biologics) is a product that is produced from living
organisms or contain components of living organisms.
• Biologic drugs include a wide variety of products derived from
human, animal, or microorganisms by using biotechnology.
• Biologic products may contain proteins that control the action of
other proteins and cellular processes, genes that control production
of vital proteins, modified human hormones, or cells that produce
substances that suppress or activate components of the immune
system.

3. Growing market of biologics

4. Antibody Structure
• Antibodies are glycoproteins. The basic functional unit of each antibody is an
immunoglobulin (Ig) monomer (containing only one Ig unit); secreted antibodies
can also be dimeric with two Ig units as with IgA, tetrameric with four Ig units like
teleost fish IgM, or pentameric with five Ig units, like mammalian IgM.
• IMMUNOGLOBULIN DOMAINS: THE Ig monomer is a “Y”-shaped molecule
that consists of four polypeptide chains; two identical heavy chains and two
identical light chains connected by disulfide bonds. Each chain is composed of
structural domains called immunoglobulin domains.

5. The antigen-binding (Fab) fragment
is a region on an antibody that binds
to antigens. It is composed of one
constant and one variable domain of
each of the heavy and the light
chain.
The fragment crystallizable region (Fc
region) is the tail region of an antibody
that interacts with cell surface receptors
called Fc receptors and some proteins of
the complement system. This property
allows antibodies to activate the immune
system.

6. Current methods of HOS Assessment of
Biopharmaceutical
Number of method used/proposed for HOS assessment:
• Chormatography:SEC/IEC/HIC
• CD,FT-IR,RAMAN
• HDX-MASS
• Fluorescence
• Light Scattering , DLS

7. Why 2D NMR ?
Advantages:
Atomistic Assignment: Biologics are complex molecules and 2D NMR allow us to separate
the signals in specific regions and gives the opportunity to assign those signals in the molecule.
Measurement reproducibility: Two dimensional NMR methods are highly reproducible
and can be implemented on different biologics with different compositions
Challenges:
Sensitivity: Sensitivity of 2D NMR is an important aspect for comparison of biologics.
Resolution(Spectral Complexity): Since biologics are macromolecules hence spectral
complexity is also a challenge for analysis .

8. NMR is Precise and Robust Approach for HOS
Assessment
• NMR can provide highly detailed fingerprint-like information about
the structure and dynamics of a biopharmaceutical drug.
NMR can find Applications in:
• Biopharmaceutical Development
• Formulation studies
• Bio-manufacturing comparability
• Quality control
• Biosimilar

9. 2D NMR of Formulated Biopharmaceuticals

10. Challenges in Applying 2D 1H-15N NMR
FINGERPRINTING TO mAbs

11. 2D 1H-13C NMR (METHYL): An Approach for mAb HOS
Fingerprints

12. Fingerprinting the Fab/Fc Fragments of mAb

13. NISTmAb,Fab and Fc Methyl Fingerprints
Here the fragments of
antibody(Fab &Fc) are sum up
together and matching up with
the whole antibody
Individual Spectrum of
antibody Fragments Fab and
Fc

14. Excipients Interference with Methyl Region
To Remove these unwanted excipients signals specific pulse sequence has been
applied.

15. Modification in Pulse sequence to remove
excipients signals
Selective pulse techniques combined with Smile-based signal subtraction can remove
excipients signal with minimal sensitivity lost.

16. Simulating structure variation in mAb by
Glycan Remodeling

17. Glycan Remodeling to study structural
changes with native mAb
In ExoGal the terminal sugar has been
trimed while in DeGly entire glycan has
been removed

18. Glycan Remodeling spectral comparison with
native mAb
Here the Linear correlation analysis has been done for modified mAbs with NISTnative
mAb

19. PCA analysis of modified mAbs with native
mAb

20. Summary
2D NMR Fingerprinting of biopharmaceutical is precise, practical and
robust method.
• NMR Spectrum can be recorded with natural abundance (No isotope labeling is required)
• By using NUS/SOFAST experiment can be done within short duration of time
• Excipients signals can be removed to enhance resolution and sensitivity
Data Analysis:
• Linear correlation analysis
• Point by point comparison
• Multivariate analysis (PCA) can be automated without interactive analysis of spectral features.