Unlike generic small-molecule drugs, which are synthesized in the laboratory and are exact copies of the reference drug, monoclonal antibodies are produced in mammalian cells due to the ability of these cells to perform post-translational modification. A drawback of expression in heterologous systems is unpredictable modifications like variable glycosylation, deamination of asparagine and glutamine, and oxidation of cysteine, methionine, or tryptophan, which result in heterogeneity in the biologic product. Unexpected modifications may affect the product's chemical properties, such as surface charge, solubility, stability, and three-dimensional structure (higher order structure or HOS). These, in turn, may affect the efficacy and safety of the treatment. For this reason, biosimilars cannot be handled as generic small-molecule drugs (and they are not considered "generic drugs"). For approval by regulatory agencies, developers must present detailed comparative studies of the biosimilar, which has to be highly similar to the reference antibody, with no clinical differences in safety, quality, and efficacy, as stated by EMA. Often, methods like UV and fluorescent spectroscopy are used for tertiary structure analysis, while Fourier Transform Infrared (FTIR) and CD spectroscopy are used for secondary structure analysis. However, X-ray crystallography and NMR spectroscopy yield much more accurate and detailed structural information. NMR spectroscopy in a time- and cost-efficient manner can be used to analyze higher order structure (HOS) of intact antibodies or other biologics. For example, a powerful application is to accurately and efficiently compare a biosimilar's higher order structure (HOS) to its originator antibody or compare production lots to ensure higher order structure batch-to-batch consistency. At SARomics Biostructures, we routinely use 2D-NMR to compare the HOS of biosimilars to originator biologics or as a verification method for the HOS of different production batches. Spectra can easily be analyzed by visual inspection and extended by different analysis methods such as peak analysis, ECHOS, or chemometric methods such as principal component analysis (PCA). The poster shown here was presented at the Protein and Antibody Engineering Summit (PEGS Europe) in Lisbon (November 14-16, 2023).

1. NMR Higher order structure (HOS) analysis of
biologics for comparison studies of batch-to-batch
consistency, biosimilars and formulation optimizations
Authors: Olof Stenström, Carl Diehl and Björn Walse
SARomics Biostructures AB, Lund, Sweden
www.saromics.com
Natural abundance NMR as a tool for HOS
comparison of biosimilars and batch control
NMR spectroscopy can be used to analyze higher order structure (HOS) of intact
antibodies or other biologics. For example, a powerful application is to accurately
and efficiently compare a biosimilar’s higher order structure to its originator anti-
body (Figure 1) or compare production lots to ensure higher order structure
batch-to-batch consistency. At SARomics Biostructures we routinely use 2D-NMR
as a method to compare the HOS of biosimilars to originator biologics or as a
verification method for HOS of different production batches. Spectra can easily be
analysed by visual inspection and can be extended by different analysis methods
such as peak analysis, ECHOS or using chemometric methods such as principal
component analysis (PCA) (see Figure 1 and Figure 2).
Contact:
Carl Diehl, NMR Services Manager, Senior Scientist, carl.diehl@saromics.com
Olof Stenström, Scientist, olof.stenstrom@saromics.com
Forced oxidation of infliximab studied
using time resolved 2D natural abundance
NMR spectroscopy
Intact or cleaved mAbs can be studied and characterized using 2D-NMR. It
should be noted that cleavage is not necessary for 2D-NMR analysis, especially
if one wishes to do HOS studies of the mAb in formulation buffer. In a joint pro-
ject, SARomics Biostructures together with Genovis AB (Lund, Sweden) used
FabALACTICA® to cleave originator and biosimilar versions of infliximab followed
by characterization and comparison using 2D-NMR. As part of this project,
forced oxidation using sodium peroxide was performed, where the oxidization
of the methionines could be detected without changing the overall higher
order structure of the cleaved Fab/Fc domains. In addition, forced oxidation of
the Fc domain were studied by time-resolved 2D-NMR with non-uniform
sampling (NUS) to reduce spacing between spectra (see Figure 3 and Figure 4).
Figure 3: 1
H-13
C 2D
NMR spectra of inflix-
imab as native Fab/
Fc (orange) and af-
ter forced oxidation
using sodium peroxide
(blue). Box indicate
region containing
methionine side cha-
ins. Box indicate
region containing
methionine side
chains.
Figure 5: 1
H-13
C 2D NMR spectra
of native hGH (orange) and un-
folded hGH at a SDS/hGH ratio
of 37 (green).
Figure 6: Zoom in on
the methionine region of
1
H-13
C 2D NMR spectra of
native hGH (orange), hGH
at SDS/hGH ratio of 4 (blue),
hGH at a SDS/hGH ratio of
37 (green) and unfolded
hGH at a SDS/hGH ratio
of 360 (red).
Figure 4: Time-
resolved oxidation
process of infliximab
Fc followed using
1
H-13
C 2D NMR spectra
at 0 (red), 2 (green)
and 16 h (blue). Top
panels show zoom in
on the methionine re-
gion (A) and oxidized
methionine region (B).
Excipient effect on HOS studied using
2D natural abundance NMR
Natural abundance 2D-NMR can be used to characterize changes to HOS due
to the influence of various excipients. SARomics Biostructures is a member of
Next- BioForm, an academic-industrial consortium focused on the formulation of
bio- logical molecules, where SARomics Biostructures contributes its expertise
in NMR spectroscopy and structural biology. As part of this consortium, the effect
of SDS on human growth hormone (hGH) was studied using a variety of physical
characterization techniques including 2D-NMR and SANS. The data clearly show
how the secondary and tertiary structure unfolds with increasing SDS concen-
tration. As part of this project, the effect of various excipients on the formulation
of biologics are studied using a toolbox of different techniques, where 2D-NMR
serves as a vital characterization technique for higher order structure (see
Figure 5 and Figure 6).
Reference: Sanchez-Fernandez,Adrian, Carl Diehl, Judith E. Houston,Anna E. Leung, James P.Tellam, Sarah E. Rogers,
Sylvain Prevost, Stefan Ulvenlund, Helen Sjögren, and Marie Wahlgren. ”An integrative toolbox to unlock the structure and
dynamics of protein–surfactant complexes.” Nanoscale Advances 2, no. 9 (2020): 4011-4023.
Figure 2: 1
H-13
C 2D NMR
spectra of NIST Fab pro-
duced in 20%-13
C-labeled
P. pastoris (orange) or
murine (green) expression
media. Boxes indicate
where differences for
methyl groups can be
detected in the spectra.
www.saromics.com
Figure 1: ECHOS analysis of 2D-NMR
spectra for quantification of similarity
between originator and biosimilar.
Box indicate how specific peak
differences can be identified from
the ECHOS analysis.