TY - JOUR

T1 - Subtle pH variation around pH 4.0 affects aggregation kinetics and aggregate characteristics of recombinant human insulin

AU - Thorlaksen, Camilla

AU - Stanciu, Adriana-Maria

AU - Neergaard, Martin Busch

AU - Jiskoot, Wim

AU - Groenning, Minna

AU - Fodera, Vito

PY - 2022

Y1 - 2022

N2 - Insulin is a biotherapeutic protein, which, depending on environmental conditions such as pH, has been shown to form a large variety of aggregates with different structures and morphologies. This work focuses on the formation and characteristics of insulin particulates, dense spherical aggregates having diameters spanning from nanometre to low-micron size. An in-depth investigation of the system is obtained by applying a broad range of techniques for particle sizing and characterisation. An interesting observation was achieved regarding the formation kinetics and aggregate characteristics of the particulates; a subtle change in the pH from pH 4.1 to pH 4.3 markedly affected the kinetics of the particulate formation and led to different particulate sizes, either nanosized or micronsized particles. Also, a clear difference between the secondary structure of the protein particulates formed at the two pH values was observed, where the nanosized particulates had an increased content of aggregated beta-structure compared to the micronsized particles. The remaining characteristics of the particles were identical for the two particulate populations. These observations highlight the importance of carefully studying the formulation design space and of knowing the impact of parameters such as pH on the aggregation to secure a drug product in control. Furthermore, the identification of particles only varying in few parameters, such as size, are considered highly valuable for studying the effect of particle features on the immunogenicity potential.

AB - Insulin is a biotherapeutic protein, which, depending on environmental conditions such as pH, has been shown to form a large variety of aggregates with different structures and morphologies. This work focuses on the formation and characteristics of insulin particulates, dense spherical aggregates having diameters spanning from nanometre to low-micron size. An in-depth investigation of the system is obtained by applying a broad range of techniques for particle sizing and characterisation. An interesting observation was achieved regarding the formation kinetics and aggregate characteristics of the particulates; a subtle change in the pH from pH 4.1 to pH 4.3 markedly affected the kinetics of the particulate formation and led to different particulate sizes, either nanosized or micronsized particles. Also, a clear difference between the secondary structure of the protein particulates formed at the two pH values was observed, where the nanosized particulates had an increased content of aggregated beta-structure compared to the micronsized particles. The remaining characteristics of the particles were identical for the two particulate populations. These observations highlight the importance of carefully studying the formulation design space and of knowing the impact of parameters such as pH on the aggregation to secure a drug product in control. Furthermore, the identification of particles only varying in few parameters, such as size, are considered highly valuable for studying the effect of particle features on the immunogenicity potential.

KW - Particulates

KW - Human insulin

KW - Aggregates

KW - Particles

KW - pH

KW - MONOCLONAL-ANTIBODY

KW - PROTEIN PARTICLES

KW - AMYLOID FIBRILS

KW - IMMUNOGENICITY

KW - PRODUCT

KW - FIBRILLATION

KW - ALPHA

U2 - 10.1016/j.ejpb.2022.09.001

DO - 10.1016/j.ejpb.2022.09.001

M3 - Journal article

C2 - 36087880

VL - 179

SP - 166

EP - 172

JO - European Journal of Pharmaceutics and Biopharmaceutics

JF - European Journal of Pharmaceutics and Biopharmaceutics

SN - 0939-6411

ER -