High-Resolution Mass Spectrometric Methods for Proteins in Lyophilized Solids

Chandrababu, Karthik Balakrishna; Kammari, Rajashekar; Chen, Yuan; Topp, Elizabeth M.

doi:10.1007/978-1-4939-8928-7_14

Cited by 1 publication

(3 citation statements)

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“…Amide hydrogen exchange, therefore, provides information on protein flexibility, conformational distributions, hydrogen-bond patterns, and structure. 3 More recently, solid-state hydrogen−deuterium exchange coupled with mass spectrometry (ssHDX-MS) has enabled a detailed analysis of protein structure and matrix interactions within amorphous solid powders produced for example by lyophilization, 4 or spray drying. 5 For solid-state exchange, vials containing a lyophilized protein formulation are placed uncapped in a sealed desiccator over a saturated salt solution of D 2 O to maintain constant D 2 O in the vapor phase.…”

Section: ■ Introductionmentioning

confidence: 99%

“…11,12 The kinetics of deuterium incorporation during ssHDX-MS are likely to report on the wider hydrogen bond network linking the protein to the amorphous solid state, which includes both intramolecular hydrogen bonds for the native protein and intermolecular hydrogen bonds to the surrounding matrix. 4 Water replacement is thought to occur in the amorphous solid state, whereby proteins become stabilized by the formation of hydrogen bonds to excipients, 13 and so deuterium can reach the protein either by local interactions between sorbed D 2 O and protein amides or by conduction of deuterons through hydrogen-bond networks within the solid. The extent of hydrogen bonding between the protein and excipients can depend on the excipient structure, molecular weight, and physical morphology of the solid.…”

Section: ■ Introductionmentioning

confidence: 99%

“…Slow exchange is thought to result from amide hydrogen atoms within stronger hydrogen bonds within the protein or to excipients, with zero exchange for the strongest structural hydrogen bonds buried within the protein core. 4 G-CSF is a widely used protein therapeutic, used to stimulate white cell proliferation after chemotherapy. 14 However, the bacterially expressed form is sensitive to lyophilization-induced stresses 15 and thus provides an ideal model for studying formulation methods and the impact of excipient addition and lyophilization on protein stability.…”

Section: ■ Introductionmentioning

confidence: 99%

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Investigation of the Solid-State Interactions in Lyophilized Human G-CSF Using Hydrogen–Deuterium Exchange Mass Spectrometry

Wood,

Kellerman,

Groves

et al. 2024

Mol. Pharmaceutics

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Hydrogen/deuterium exchange mass spectrometry (HDX-MS) previously elucidated the interactions between excipients and proteins for liquid granulocyte colony stimulating factor (G-CSF) formulations, confirming predictions made using computational structure docking. More recently, solid-state HDX mass spectrometry (ssHDX-MS) was developed for proteins in the lyophilized state. Deuterium uptake in ssHDX-MS has been shown for various proteins, including monoclonal antibodies, to be highly correlated with storage stability, as measured by protein aggregation and chemical degradation. As G-CSF is known to lose activity through aggregation upon lyophilization, we applied the ssHDX-MS method with peptide mapping to four different lyophilized formulations of G-CSF to compare the impact of three excipients on local structure and exchange dynamics. HDX at 22 °C was confirmed to correlate well with the monomer content remaining after lyophilization and storage at −20 °C, with sucrose providing the greatest protection, and then phenylalanine, mannitol, and no excipient leading to progressively less protection. Storage at 45 °C led to little difference in final monomer content among the formulations, and so there was no discernible relationship with total deuterium uptake on ssHDX. Incubation at 45 °C may have led to a structural conformation and/or aggregation mechanism no longer probed by HDX at 22 °C. Such a conformational change was observed previously at 37 °C for liquid-formulated G-CSF using NMR. Peptide mapping revealed that tolerance to lyophilization and −20 °C storage was linked to increased stability in the small helix, loop AB, helix C, and loop CD. LC-MS HDX and NMR had previously linked loop AB and loop CD to the formation of a native-like state (N*) prior to aggregation in liquid formulations, suggesting a similar structural basis for G-CSF aggregation in the liquid and solid states.

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Section: ■ Introductionmentioning

confidence: 99%