PO Q1 2021 - 16

CONTRACT RESEARCH

and from Pseudomonas.4 Proteins with identical primary structure
can exhibit different therapeutic performance on account of
their secondary, tertiary or quaternary structure and therefore be
rigorously compared to confirm comparability.

complement the previously acquired results, improving the totality
of evidence of similarity by directly comparing the composition
of secondary structural elements and demonstrating the high
conservation of secondary structure. The percentage of beta-turn,
alpha helix, unordered and beta-sheet structure in each type of
CRM197 is closely similar and well-matched to levels observed via
crystal structure analysis of the diphtheria toxin.

Observation and conservation of a protein's secondary structural
components, such as alpha-helix and beta-sheet is critical to the
demonstration of bioequivalence. The tertiary structure of a protein
is its overall three-dimensional shape which is primarily defined by
non-covalent bonding between the R groups of the constituent
amino acids. However, disulfide bonds also have an important
impact, not least because of their significant strength relative to other
contributing bonds. As already discussed, these bonds are particularly
relevant to the functionality of CRM197. In the preliminary studies the
secondary structure of the CRM197 samples was characterized by
circular dichroism (CD), a widely used technique that is most effective
for simple, dilute samples and tertiary structure was characterized
using three different techniques: second derivative UV analysis;
intrinsic fluorescence; and extrinsic fluorescence (which involves the
use of an added fluorophore). All these early analyses supported a
claim of comparability in the EcoCRM® and provided preliminary
evidence that it is correctly folded.

Working Towards an
Optimized Formulation
As a result of successfully applying MMS to provide evidence
of similarity in secondary structure the FinaBio team decided
to incorporate the technique in formulation studies. Unlike CD,
MMS measures consistently and reliably across a wide range of
concentrations, and in the presence of excipients, a defining feature
of the technology that brings considerable value in formulation
studies. The preceding results also highlight it as the more informative
technique for the characterization of secondary structure, with the
capability to more precisely quantify secondary structural elements.5
As part of the development work, stability trials were carried out
with two different formulations, made up with 'Old' and 'New' buffers.
Samples were held for up to 12 weeks at two temperatures (20°C and
4°C) with analysis carried out after one, two, four and twelve weeks. A
freshly thawed sample was used for reference.

Satisfactorily confirming biosimilarity can be particularly challenging
for biotherapeutic proteins and it is common to apply a battery of
orthogonal biophysical tests to robustly identify any detectable
difference. In further work, an additional analytical technique,
Microfluidic Modulation Spectroscopy, was therefore applied to
extend the evidence base for comparability between the secondary
structure of the EcoCRM® and one of the existing commercial sources
of CRM197.

Figure 2 shows the MMS data gathered over the course of the stability
trial. Replicates for each sample of the new formulation are very
consistent demonstrating the high repeatability of the technique once
the effect of concentration, which varies slightly across the sample
set, is taken into account. Absolute spectra (Figure 2a), corrected for
the effect of concentration, overlay very closely indicating highly
consistent secondary structure under all storage conditions.

MMS is a technique that generates drift-free, background
compensated differential infra-red (IR) absorption scans of the Amide
I band by modulating the sample with a matching buffer through the
detector during sample acquisition. This band is associated with the
C=O stretch vibration of peptide linkages along the protein backbone
and is sensitive to changes in secondary structure.

Second derivative plots, which help to highlight similarity or difference
in a set of samples, were produced for both formulations using the
instrument data analysis software (AQS3delta - Figure 2b). These data
clearly indicate differences with respect to stability over the 12-week
period. Area of Overlap analysis, a numerical analysis of the extent to
which the area under one second derivative curve overlaps with that
of another, was carried out to quantify this difference and was used to
produce plots of dissimilarity (Figure 2c). These plots show that in the
old buffer the CRM197 exhibits differences in structural change after
1 week at room temperature (orange bars) and after 2 weeks at 4°C
(gold bars). In contrast, in the new buffer, structural change was not
observed until the 12-week time point. In the new formulation the
protein is only around 1% dissimilar from the control after 12 weeks
at room temperature (dark blue bars).

Figure 1 shows the percent composition of secondary structure
determined by MMS analyses of the two sources of CRM197. These data
Figure 1. Comparative structural data for two sources of
CRM197 provide further evidence of their equivalence.

The spectral scans were further processed to directly compare
the amount of each element of sub-structure in the stored new
formulation and freshly thawed control samples. This analysis
confirmed that the measured elements of secondary structure - betasheet, beta-turn, alpha-helix and unordered - were preserved in the
new formulation for the duration of the trial, with variability across
Pharmaceutical Outsourcing |

16

| January/February/March 2021



PO Q1 2021

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