Monoclonal antibodies are one of the fastest growing classes of pharmaceutics. Although they
possess an almost completely human aminoacidic sequence, special attention needs to be
paid to limit adverse immune responses, which are, due to invasive application, most often
caused by aggregates of different sizes. To ensure efficacy, safety, and quality of biological
drug products until the end of their shelf life, it is therefore essential to find conditions that
efficiently slow down aggregation already in the early development. Evaluation and
optimization of long-term stability is thus a crucial step in development of biologics, which is
still long-lasting and introduces substantial risk to the development. To date it hasn’t been
unambiguously shown that increasing thermodynamic and colloidal stability, a primary
strategy in the recent years, really leads to lower aggregation rate. On the other hand, with
aim to accelerate development, aggregate increase in different formulations is being evaluated
at higher temperatures, which doesn’t necessarily reflect changes until the end of shelf-life at
storage temperature, usually three years at 5 °C.
In this work, I present several approaches for shortening time needed for aggregation
assessment and, thus, accelerating formulation optimization aimed specifically at slowing
down aggregation at storage conditions. For the group of antibodies used in oncology,
immunology, rheumatology, for therapy of osteoporosis, and blood diseases, I managed to
successfully describe aggregation data from wide temperature and concentration range by
applying a kinetic aggregation mechanism comprised of two pathways, low- and
high-temperature pathway. Additionally, model accurately forecasts aggregate fractions up to
three years based on data obtained in much shorter period.
Kinetic aggregation mechanism only implies existence of two distinct, temperature-dependent
pathways. Therefore, I isolated several aggregate fractions of the selected antibody mAb1.
Analysis of antibody’s chemical modifications in separate fractions with cIEF and PepMap-MS
methods and analysis of activation energy values revealed that aggregates formed at
temperature typical for low-temperature aggregation importantly differ from aggregates formed
at temperature typical for high-temperature aggregation. Besides differences in oxidation and
deamidation level, these results, with aid of available crystal structure of human IgG antibody,
indicate that high-temperature aggregation is accompanied by the partial antibody unfolding.
Analysis of aggregation on two pathways requires extensive study for every assessed
formulation, which is highly impractical in pharmaceutical development. Simplified model that
considers aggregation as a (pseudo) first order kinetic reaction enables large acceleration and
simplification in comparing aggregation susceptibility of formulations, when target
concentration is known beforehand. Based on data from at least three temperatures, at which
high-temperature pathways do not contribute to total aggregation, we can calculate long-term
aggregation rate at any temperature with satisfactory accuracy. If we choose low enough
temperature, we can directly compare formulations between themselves and thus in a most
efficient way narrow down set of excipients and conditions successfully decreasing
aggregation at storage temperature.
Apparent standard Gibbs free energy of denaturation obtained by analysis of antibody
chemical denaturation is a parameter of thermodynamic stability, which is a good indicator of
antibody’s aggregation propensity. A constructed aggregation phase space revealed that we
can effectively slow down aggregation of antibodies with low thermodynamic stability by its
increase. This is explained by decreasing pool of initial aggregating intermediate. On the other
hand, for antibodies with high thermodynamic stability, its value primarily affects kinetic
constant for formation of this intermediate, with the effect being much weaker in comparison.
Nevertheless, analysis of thermal denaturation offers a useful information for the development
of biopharmaceuticals. Namely, temperature, up to which aggregation through
high-temperature pathway doesn’t contribute substantially, is approximately 15 or 25 °C lower
than the thermal denaturation transition temperatures of CH2 and Fab domains, respectively.
Strategy combining kinetic and thermodynamic analysis not only improves efficiency of
biologic drug product development and production but contributes to a better understanding
of molecular mechanisms of aggregation as well.
|
