TY - JOUR

T1 - Combination of high throughput and structural screening to assess protein stability – A screening perspective

AU - Pohl, Christin

AU - Mahapatra, Sujata

AU - Kulakova, Alina

AU - Streicher, Werner

AU - Peters, Günther H.J.

AU - Nørgaard, Allan

AU - Harris, Pernille

N1 - Funding Information: The synchrotron SAXS data was collected at beamline P12 operated by EMBL Hamburg at the Petra II storage ring (DESY, Hamburg, Germany. For local support we thank Melissa Gr?wert, Stefano Da Vela, Andrey Gruzinov, Karen Manalastas (all: EMBL, Hamburg). All data is publicly available from the PIPPI database https://pippi-data.kemi.dtu.dk/, CP wrote the manuscript with support from SM, AN and PH. AK performed and analyzed the SEC-MALS experiments. PH, AN, WS and GHJP planned and supervised the study. CP conducted, analyzed and interpreted the experiments on TLL. SM conducted and analyzed the experiments on RML. CP and SM interpreted the experiments on RML. All authors corrected and approved the final manuscript. This work was funded by European Union's Horizon 2020 research and innovation program (grant agreement no. 675074). We thank the Danish Agency for Science, Technology, and Innovation for funding the instrument center DanScatt. Funding Information: We thank the Danish Agency for Science, Technology, and Innovation for funding the instrument center DanScatt. Funding Information: This work was funded by European Union’s Horizon 2020 research and innovation program (grant agreement no. 675074). Publisher Copyright: © 2021 Elsevier B.V.

PY - 2022/2

Y1 - 2022/2

N2 - High throughput screening for measuring the stability of industrially relevant proteins and their variants is necessary for quality assessment in the development process. Advances in automation, measurement time and sample consumption for many techniques allow rapid measurements with minimal amount of protein. However, many methods include automated data analysis, potentially neglecting important aspects of the protein's behavior in certain conditions. In this study we implement small angle X-ray scattering (SAXS), typically not used to assess protein behavior in industrial screening, in a high throughput screening workflow to address problems of contradicting results and reproducibility among different high throughput methods. As a case study we use the lipases of Thermomyces lanuginosus and Rhizomucor miehei, widely used industrial biocatalysts. We show that even the initial analysis of the SAXS data without performing any time-consuming modelling provide valuable information on interparticle interactions. We conclude that recent advances in automation and data processing, have enabled SAXS to be used more widely as a tool to gain in-depth knowledge highly useful for protein formulation development. This is especially relevant in light of increasing accessibility to SAXS due to the commercial availability of benchtop instruments.

AB - High throughput screening for measuring the stability of industrially relevant proteins and their variants is necessary for quality assessment in the development process. Advances in automation, measurement time and sample consumption for many techniques allow rapid measurements with minimal amount of protein. However, many methods include automated data analysis, potentially neglecting important aspects of the protein's behavior in certain conditions. In this study we implement small angle X-ray scattering (SAXS), typically not used to assess protein behavior in industrial screening, in a high throughput screening workflow to address problems of contradicting results and reproducibility among different high throughput methods. As a case study we use the lipases of Thermomyces lanuginosus and Rhizomucor miehei, widely used industrial biocatalysts. We show that even the initial analysis of the SAXS data without performing any time-consuming modelling provide valuable information on interparticle interactions. We conclude that recent advances in automation and data processing, have enabled SAXS to be used more widely as a tool to gain in-depth knowledge highly useful for protein formulation development. This is especially relevant in light of increasing accessibility to SAXS due to the commercial availability of benchtop instruments.

KW - Drug screening

KW - High throughput screening

KW - Protein aggregation

KW - Protein engineering

KW - Protein stability

KW - Protein–protein interaction

KW - Small-angle X-ray scattering (SAXS)

U2 - 10.1016/j.ejpb.2021.08.018

DO - 10.1016/j.ejpb.2021.08.018

M3 - Journal article

C2 - 34826593

AN - SCOPUS:85121711133

VL - 171

SP - 1

EP - 10

JO - European Journal of Pharmaceutics and Biopharmaceutics

JF - European Journal of Pharmaceutics and Biopharmaceutics

SN - 0939-6411

ER -