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Microfluidic Modulation Spectroscopy for Protein Characterization A Case Study with EcoCRM, CRM197 Carrier Protein

November 6, 2020

Andrew Lees, Min-Ju Chang and Libo Wang

The biophysical characteristics of biological drug products must be monitored from development through formulation and commercialization. Rapid screening of multiple parameters facilitates characterization under a wide range of conditions. This biophysical characterization is also useful in establishing the comparability of biosimilars. RedShift Bioanalytics has developed an instrument which uses microfluidic modulation spectroscopy to monitor five key biophysical properties of proteins: aggregation, quantitation, stability, similarity and structure. The instrument uses mid-infrared absorption spectroscopy and rapidly produces differential IR scans which measure the amide I band (the C=O stretching vibration of peptide backbone linkages).The amide I band is highly sensitive to changes in the secondary structure of the protein. The spectrophotometric technology and signal processing software achieves significant increases insensitivity, dynamic range, and accuracy for determination of protein secondary structure relative to conventional mid-IR and far-UV CD techniques. The instrument works well for comparing samples across a wide dynamic range of concentrations and buffer conditions. In this study, we evaluated the biophysical properties of CRM197, a widely used conjugate vaccine carrier protein, produced by two different bacterial systems: EcoCRM® (Fina Biosolutions LLC, Rockville, MD)which is expressed as a soluble, intracellular, properly-folded protein in E. coli and CRM197 expressed in the periplasm of Pseudomonas fluorescence (Pfenex, Inc., San Diego). We compared the properties ofCRM197 from the two sources. Both the E. coli and Pseudomonas expressed CRM197. To further the formulation development, we also evaluated Fina Biosolutions’ EcoCRM® stability under stress.

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