High-Throughput Screening for Colloidal Stability of Peptide Formulations Using Dynamic and Static Light Scattering

Abstract

Selection of an appropriate formulation to stabilize therapeutic proteins against aggregation is one of the most challenging tasks in early-stage drug product development. The amount of aggregates is more difficult to quantify in the case of peptides due to their small molecular size. Here, we investigated the suitability of diffusion self-interaction parameters (k D) and osmotic second virial coefficients (B 22) for high-throughput (HT) screening of peptide formulations regarding their aggregation risk. These parameters were compared to the effect of thermal stress on colloidal stability. The formulation matrix comprised six buffering systems at two selected pH values, four tonicity agents, and a common preservative. The results revealed that electrostatic interactions are the main driver to control colloidal stability. Preferred formulations consisted of acetate and succinate buffer at pH 4.5 combined with glycerol or mannitol and optional m-cresol. k D proved to be a suitable surrogate for B 22 as an indicator of high colloidal stability in the case of peptides as was previously described for globular proteins and antibodies. Formulation assessment solely based on k D obtained by HT methods offers important insights into the optimization of colloidal stability during the early development of peptide-based liquid formulations and can be performed with a limited amount of peptide (∼360 mg).

Impact of process stress on protein stability in highly-loaded solid protein/PEG formulations from small-scale melt extrusion

“…Most of the analytical methods applied during protein formulation development studies focus on the investigation and characterization of proteins in solution or lyophilized proteins after their reconstitution. Analytical characterization methods for liquid protein formulations include: (i) RP-HPLC for the analysis of degradation products and chemical stability, (ii) size exclusion chromatography (SEC) for monitoring the loss of protein monomers as an indication of aggregate formation, (iii) circular dichroism (CD) and nuclear magnetic resonance (NMR) to probe conformational stability and (iv) light scattering methods for estimating the colloidal stability of protein formulations and monitoring the appearance of sub-visible particles ( Capelle et al, 2007 ; Chaudhuri et al, 2014 ; Dauer et al, 2021a ; Dauer et al, 2021b; Bhirde et al, 2020 ; Ma et al, 2020 ).…”

Long-Term Stability Prediction for Developability Assessment of Biopharmaceutics Using Advanced Kinetic Modeling

“…Three compositions were selected based on (i) preliminary chemical stability data as well as (ii) an automated formulation screening with focus on aggregation and physical stability, as exemplified in Refs. [20,21,45]. Peptide concentrations of 0.1, 0.5, and 2.5 mg/mL appeared suitable for the intended dosing scheme of phase 1 clinical trials.…”

“…Two very different tonicity adjusting agents were explored: (i) glycerol as a non-ionic polyol that does not increase ionic strength above the level imposed by the acetate buffer (formulation F1) and (ii) NaCl as the most common ionic tonicity agent which significantly increases the ionic strength on top of the acetate buffer (formulation F2). Differences in ionic strengths may significantly affect chemical degradation pathways and influence physical stability properties [21]. A further formulation (F3) contains low levels of polysorbate 20 (PS20) as a potential stabilizer for long-term physical stability that may be needed to reduce aggregation risk under mechanical stress (e.g., aggregation at liquid-air or liquidglass interfaces).…”

“…[5,20]. As part of this developability assessment, the best candidate molecules are evaluated in an extended physico-chemical property profiling that includes analysis of pH-dependent degradation in buffered solutions and a robotic formulation screening focused on physical stability [21]. Formulations with low aggregation risk are then subjected to chemical stability testing under accelerated conditions (40 • C for 4 weeks).…”

Effects of Monovalent Salt on Protein-Protein Interactions of Dilute and Concentrated Monoclonal Antibody Formulations

“…These interactions can be either attractive or repulsive in nature [ 29 , 30 ]; therefore, they diminish or enhance the interaction between two protein molecules. When considering proteins as colloid particles, the tendency of proteins to stay in their monomeric form is typically referred to as their colloidal stability, and it is dominated by the net balance between repulsive and attractive protein-protein interactions (PPI) [ 31 ].…”