Several protein stabilizers including amino acids (arginine, histidine, glycine) and sugars (trehalose) are being successfully applied in improving mAb formulations in addition to the excessively used surfactants such as polysorbates ( 9, 11).
#Dsc dls panel could not be identified free
The preferential exclusion of the stabilizing solutes from the protein surface increases the free energy of the native state of the proteins ( 10). This enables the small molecule osmolytes to act as protectants by preferential exclusion from the protein surface, rendering enhanced stability to the macromolecule. As per Arakawa et al., the mechanism of the effect of the protein stabilizing osmolytes can be elucidated via preferential hydration of the protein surface due to the exclusion of solutes as well as the restructuring of water molecules around the protein molecule in solution. One of the mechanisms by which these small molecule osmolytes act as protectants is preferential exclusion from the protein surface, rendering enhanced stability to the macromolecule ( 8, 9). One pragmatic strategy to minimize aggregation is the recruitment of compatible solutes that enhance the protein surface hydration and prevent unfolding. Thermal aggregation of IgG proteins can be triggered by partial or complete unfolding, thereby leading to formation of soluble oligomers and eventually insoluble aggregates through interactions between the oligomeric species ( 7). As a result, aggregation of a drug product (DP) has a significant impact on shelf life of the biotherapeutic, and all biopharmaceutical manufacturers engage in thorough biophysical characterization of aggregates. Studies reveal that formation of HMWs not only results in a significant loss of biological activity and efficacy but can also potentially lead to toxicity and adverse immunogenic reactions post patient administration, raising questions about product safety ( 4– 6).
Aggregation, i.e., formation of high molecular weight species (HMWs) of IgG therapeutic proteins, is widely considered to be a critical quality attribute (CQA)( 3). Clinical application necessitates development of stable formulations, as these proteins are susceptible to various degradation pathways including the propensity of mAbs to aggregate at high concentration, elevated temperatures, and varying pH ( 2). Our data support the prospect of using these osmolytes as successful excipients for mAb formulations.Ī majority of the marketed biotherapeutics used to treat life-threatening diseases, such as cancer and immune disorders, are monoclonal antibodies (mAbs) owing to their binding with high specificity to their targets ( 1). Sarcosine emerged as the most successful osmolyte rendering highest degree of protection against aggregation.
Our results rank the osmolytes’ stabilizing trend to be sarcosine > betaine > hydroxyectoine > ectoine. No significant impact of osmolyte addition was observed on protein structure, on comparative Fc receptor (FcRn) binding, and on biocompatibility as per our hemolytic assay. A variety of analytical tools have been used for monitoring the impact, dynamic light scattering (DLS) for colloidal stability, Fourier transform infrared (FTIR) spectroscopy and fluorescence spectroscopy for conformational stability and the higher order structure (HOS), and differential scanning calorimetry (DSC) for thermal stability. Experimentation has been performed on two IgG1 mAbs via accelerated stability studies. In this paper, we explore potential use of naturally occurring osmolytes such as betaine, sarcosine, ectoine, and hydroxyectoine for reducing aggregation of mAb therapeutics. Osmolytes such as trehalose, sucrose, and glycine are widely used. One of the most commonly used strategy to overcome protein aggregation is addition of excipients to the formulation. Monoclonal antibodies (mAbs), while incredibly successful, are prone to a variety of degradation pathways, the most significant of which is aggregation.