Abstract
Assessing the physical stability of proteins is one of the most important challenges in the development, manufacture, and formulation of biotherapeutics. Here, we describe a method for combining and automating circular dichroism and intrinsic protein fluorescence spectroscopy. By robotically injecting samples from a 96-well plate into an optically compliant capillary flow cell, complementary information about the secondary and tertiary structural state of a protein can be collected in an unattended manner from considerably reduced volumes of sample compared to conventional techniques. We demonstrate the accuracy and reproducibility of this method. Furthermore, we show how structural screening can be used to monitor unfolding of proteins in two case studies using (i) a chaotropic denaturant (urea) and (ii) low-pH buffers used for monoclonal antibody (mAb) purification during Protein A chromatography.
Highlights
The majority of biologics are recombinant proteins produced by engineered microbial, plant, or animal cells, and processes for their manufacture rely on harmonious integration of a complex series of upstream and downstream operations.[1]
Asymmetric absorption of the peptide backbone in the far-UV region provides an excellent means of measuring changes in the total secondary structure of a protein, while absorption in the near-UV region arising from aromatic amino acids provides a good indicator of tertiary structure changes
To test the capillary-based Circular dichroism (CD)/fluorescence measurement system (Figure 1) for its suitability in automated bioprocess development work, calibration data were collected from samples of 1 mg/mL CSA (Figure 2A) and 0.1 mg/mL bovine serum albumin (BSA) (Figure 2B)
Summary
Article (ideally in the same cuvette for CD) with cleaning between measurements, performing spectral analyses on many samples can be time-consuming. Collection of full CD and fluorescence spectra using automated triple air-segmented sample delivery was subsequently evaluated by loading varying concentrations of BSA and mAb in the ht-caCD/F system and pausing flow for scanning routines. This entailed preparing multiwell plates containing repeated sequences of buffer blank, protein sample, and 2% (v/v) Hellmanex cleaning solution and automatically collecting CD and fluorescence spectra from 40 μL samples loaded independently three times. The spectral changes recorded here are in keeping with two phase structural rearrangements previously observed for immunoglobins exposed to low pH,[47,48] with onset of unfolding occurring below pH 6 and severe distortion of structure evident below pH 3
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