1
Unfolding and Aggregation of mAbs
Application Note NT-PR-005
Analysis of formulation-dependent colloidal and
conformational stability of monoclonal antibodies
Franziska Söltl
1
, Jonathan Derix
1
, Patrick Garidel
2
, Michaela Blech
2
and Dennis Breitsprecher
1
1
NanoTemper Technologies GmbH, Munich, Germany
2
Boehringer Ingelheim Pharma GmbH & Co. KG, Global Bioprocess & Pharmaceutical
Development, Global Formulation Development, Biberach, Germany
Abstract
The growing number of biological drugs such
as monoclonal antibodies (mAbs), as well as
the wealth of heterogeneity between mAb
variants requires a thorough development
process to maximize mAbs compliance with
regulation. Therefore, biophysical analytical
methods are required already at early stages of
the development process to guide and
streamline further antibody processing and to
predict antibody developability.
In this case study, we demonstrate how the
Prometheus NT.48 can be used to predict long-
term mAb stability in a formulation screen by
simultaneous quantification of both,
conformational and colloidal stability of
biologicals in thermal gradients.
Introduction
Monoclonal antibodies (mAbs) constitute the
majority of therapeutic biologicals today. Owed to
their high specificity and potency, they are used to
treat a number of diseases, ranging from different
cancer types to autoimmune defects. Novel protein
engineering approaches lead to a growing number
of therapeutic mAbs, which can additionally be
modified to be bispecific, conjugated with other
biologicals or modified with small molecule drugs.
The conformational and colloidal stability of
antibodies are key parameters to predict their
stability and also developability, since they affect
the long-term storage stability which is critical for
Figure 1: Relation between conformational and colloidal
stability of antibodies. (A) Equilibrium and non-equilibrium
states between folded and unfolded antibodies and aggregates.
(B) Schematic plot showing the consequence of strong
aggregation of the unfolded state. Irreversible aggregation leads
to an accumulation of aggregates over time, eventually resulting
in complete aggregation of the mAb. (C) Schematic
representation of directed unfolding and aggregation of
antibodies in a thermal gradient.