Abstract
High-throughput light scattering instruments are widely used in screening of biopharmaceutical formulations and can be easily incorporated into processes by utilizing multi-well plate formats. High-throughput plate readers are helpful tools to assess the aggregation tendency and colloidal stability of biological drug candidates based on the diffusion self-interaction parameter (kD). However, plate readers evoke issues about the precision and variability of determined data. In this article, we report about the statistical evaluation of intra- and inter-plate variability (384-well plates) for the kD analysis of protein and peptide solutions. ANOVA revealed no significant differences between the runs. In conclusion, the reliability and precision of kD was dependent on the plate position of the sample replicates and kD value. Positive kD values (57.0 mL/g, coefficients of variation (CV) 8.9%) showed a lower variability compared to negative kD values (−14.8 mL/g, CV 13.4%). The variability of kD was not reduced using more data points (120 vs. 30). A kD analysis exclusively based on center wells showed a lower CV (<2%) compared to edge wells (5–12%) or a combination of edge and center wells (2–5%). We present plate designs for kD analysis within the early formulation development, screening up to 20 formulations consuming less than 50 mg of active pharmaceutical ingredient (API).
Highlights
Biopharmaceuticals play an increasingly important role in the treatment of various diseases, and hundreds of these biopharmaceuticals are in the clinical development pipeline [1].The development of stable liquid protein formulations is often challenging and time- and material-consuming [2]
We investigated the need of an adequate outlier test and the number of minimum concentration steps for a reliable kD analysis
No L6876), acetic acid, ortho-phosphoric acid 85%, trifluoroacetic acid (TFA), acetonitrile, and DMSO were obtained from Merck Chemicals GmbH (Darmstadt, Germany)
Summary
Biopharmaceuticals play an increasingly important role in the treatment of various diseases, and hundreds of these biopharmaceuticals are in the clinical development pipeline [1].The development of stable liquid protein formulations is often challenging and time- and material-consuming [2]. The most challenging task within the early development of protein-based therapeutics is their limited stability due to a labile nature of structural integrity. This aspect evokes a high susceptibility to physical and chemical degradation at any point throughout the lifecycle of a protein and even during administration of its liquid solution [4]. In this context, protein aggregation is one of the most common physical instabilities. Colloidal stability results from protein–protein interactions and is an important factor for protein aggregation [2]
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