Redefining Efficiency in Biotherapeutic Protein Analysis - PerkinElmer eBook - 23

Redefining Efficiency in Biotherapeutic Protein Analysis * Intact Mass Analysis of Complex Therapeutic Proteins under Native Conditions

inherent to the nature of the molecule-due to
things such as glycosylation-requires significant
and robust characterization, not only during
discovery and research but throughout the
drug-development process, as well as during
quality control (QC) exercises and lot-release
testing.
Antibody-drug conjugates (ADCs) are a newer
class of antibody-based biopharmaceuticals that
entered the market in 2001. They are complex
molecules composed of an antibody targeting a
certain tumor marker linked to a small-molecule
cytotoxin. There are different chemistries available to attach the cytotoxin to the antibody, and
the coupling results in a distribution with varying
numbers of drugs attached to a single antibody
molecule, requiring the need to monitor the
drug-to-antibody ratio (DAR).
There are a vast number of analytical
methodologies that can be applied during the
development process of mAbs and ADCs, from
chromatographic techniques such as sizeexclusion chromatography (SEC), ion-exchange
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chromatography (IEC), traditional reversed-phase
(RP) liquid chromatography (LC), and capillary
electrophoresis (CE); to enzyme-linked immunosorbent assays (ELISAs). High-resolution
accurate-mass-mass spectrometry (HRAM-MS)
can be added to several of these chromatographic
techniques to provide increased confidence in
detection.

Intact Mass Analysis under
Native Conditions
The analysis of protein therapeutics at the intact
level can involve sample preparation, such as
treatment with glycosidases for mass determination of the plain protein, or in the case of ADCs, it
may be necessary to simplify the sample significantly for the accurate determination of the DAR.
Additionally, depending on the choice of
chromatographic separation conditions, the
three-dimensional protein structure of a mAb
is either preserved or disrupted:
* Use of aqueous buffers at near-neutral pH
results in the detection of fewer and lower

charge state; these results appear at the highermass end of the m/z scale (Figures 2A and 2B).
This pH fosters native or native-like conditions.
* Organic solvents, such as acetonitrile in the
presence of acids (e.g., formic acid)-typically
used in reversed-phase chromatography-
disrupt protein folding. The structure opens up
and provides a larger surface, enabling more
protons to attach, leading to the detection of
extra and higher charge states, and therefore,
results appear on the lower end of the m/z
scale. This is an example of intact mass analysis
under denaturing conditions.
Analysis under native conditions involves
instrumental requirements regarding massdetection capabilities towards 8,000 m/z to
capture the entire charge envelope, as well as
capabilities to provide sufficient desolvation,
which is more challenging when electrospraying
purely aqueous solvents without any acid
additive. Moreover, the efficient transfer of the
lower-charged ions of large proteins, or even
protein complexes, requires optimization.
For research use only. Not for use in diagnostic procedures.

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Redefining Efficiency in Biotherapeutic Protein Analysis - PerkinElmer eBook

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