Molecular Thermodynamics of Protein Systems

Curtis, Robin

doi:10.1016/b978-0-12-811556-5.00011-9

Cited by 1 publication

(1 citation statement)

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“…Dynamic Light Scattering (DLS) is commonly used to measure protein-protein interactions (PPI) or protein reversible self association 76 in terms of the diffusion interaction parameter (kD) , which is a key parameter for determining concentrated formulation properties such as viscosity, opalescence, or phase separation tendency. 76,77 The sample concentrations for the MS experiments are much lower than those typically used in formulation (> 50 mg/mL) 78 , yet molecular-level data on structural stability should be able to inform on protein-protein associations occurring at higher concentrations. To bridge between the lower concentrations used for MS to those in the formulated product, we have measured kD for each buffer of interest (Figure 4, Table S3).…”

Section: Can We Use Native Ms To Examine Aggregation As a Function Of...mentioning

confidence: 99%

Hybrid mass spectrometry applied across the production of antibody biotherapeutics.

Christofi,

O'Hanlon,

Curtis

et al. 2024

Preprint

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Post expression from the host cell, a bio-therapeutic will be subjected to several downstream processing steps prior to final formulation. Analysis of the product under various buffer conditions at each stage is desirable to show that the integrity of the sample is maintained and to monitor any potential product loss. In this study we examine how varying buffer conditions during downstream processing impact the structural integrity of a biopharmaceutical product, with a specific focus on the model IgG1 antibody, mAb4, provided by FUJIFILM Diosynth Biotechnologies. Flexibility, stability, aggregation propensity, and bulk properties were evaluated across the buffers of four sample points, including perfusion media, purification stages, and formulation buffer. Comparisons with Herceptin, an extensively studied IgG1 antibody, were conducted in a mass spectrometry-compatible buffer. Although mAb4 and Herceptin present with similar charge state distributions (CSD) in native mass spectrometry (MS) experiments, they exhibit distinct unfolding patterns during the activated ion mobility-mass spectrometry (aIM-MS) and differential scanning fluorimetry (DSF). The greater structural stability and aggregation onset temperature (Tagg) observed in Herceptin, are primarily attributed to the heavier glycosylation and kappa-class light chains present in Herceptin compared to the lambda-class light chains in mAb4. Hydrogen deuterium exchange mass spectrometry (HDX-MS) revealed that while mAb4 experiences substantial structural changes during purification, characterized by high flexibility, a low melting temperature (Tm), and prevalent repulsive protein-protein interactions, it eventually transitions to a highly compact and stable structure in buffers with higher salt concentrations and during formulation. Notably, in the formulated environment, the third constant domain (CH3) of the heavy chain retains flexibility and is identified as a region of interest for aggregation formation. Here we showcase the integration of MS and orthogonal techniques to attain comprehensive information which can be utilized early in the development stage to aid in decision-making regarding targeted mutations or to guide the design space of bioprocesses and formulation choices.

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