Protein-protein interactions in high protein concentrations

Sujata Mahapatra*

*Corresponding author for this work

Research output: Book/ReportPh.D. thesis

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Protein-based therapeutics are increasingly important due to their high specificity, potency and low toxicity. In the drug product, the protein is expected to remain chemically and physically stable over time, as degradation and aggregation can decrease the efficacy of the protein-drug, or be regarded as potentially toxic. High protein concentrations may lead to self-association, phase separation, high viscosity, opalescence, gel formation or increased propensity for protein particle formation. In the PIPPI Horizon2020 ETN project, different scientific fields are combined to systematically investigate different formulation conditions and map the proteins´ physicochemical properties, the colloidal and conformational protein stability, to assess the intermolecular interactions for the investigated proteins and also, where possible to generate molecular models. To achieve this, all the data from the PhD students in the PIPPI project is collated in a large database to make it publicly accessible for the scientific community.
This PhD project is one of the PhD projects in the PIPPI consortium. The thesis is divided into three parts: In the first part, seven proteins were chosen from PIPPI protein library to perform stability studies, from which there were five monoclonal antibodies (mAbs), a human serum albumin and one lipase. Each protein was extensively studied under different physicochemical conditions using high-throughput techniques like nano differential scanning fluorimetry (nanoDSF) and isothermal chemical denaturation (ICD). These studies were used to choose conditions to perform structural and interaction studies using small angle X-ray scattering (SAXS). In the continuation of the first part, the molecular basis for protein-protein interactions at high concentration were studied by measuring the static structure factor in the second part. Four mAbs that could be obtained at high concentrations were chosen. SAXS and static light scattering (SLS) were performed to determine the structure factor, the second virial coefficient and thereby the nature of the intermolecular interactions. Further, the effect of NaCl was also studied for one of the mAbs. According to experimental data, all four mAbs were repulsive in nature and presence of NaCl screened the repulsion. The experimental behavior could be captured by two simplified theoretical hard sphere models containing long-range repulsion and short range attraction potentials. In the third part, interaction and ion binding studies were performed for two mAbs using SAXS, SLS and coarse-grained modeling. The SAXS and SLS data indicated that the antibodies behave differently in presence of NaCl. One mAb (PPI03) has repulsive interaction although increasing ionic strength screens part of the electrostatic repulsion, while the other mAb (PPI13) has additional attractive interactions even at low NaCl concentration. To study the Cl- ion binding, metropolis monte carlo (MC) simulations were performed on the SAXS derived rigid body models. The MC simulation was used in an attempt to explain the different behavior in terms of charges and ion distributions around the mAbs by creating iso-density maps of Cl- ions. The study showed that PPI03 has more positive patches compared to PPI13 due to which Cl- ions accumulate more on PPI03. Moreover, this mechanism can be responsible for repulsion in case of PPI03 in presence of NaCl.
Original languageEnglish
Place of PublicationKgs. Lyngby
PublisherTechnical University of Denmark
Number of pages138
Publication statusPublished - 2019


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