Quantitative Analysis of Peptide–Matrix Interactions in Lyophilized Solids Using Photolytic Labeling

Peptide–matrix interactions in lyophilized solids were explored using photolytic labeling with reversed phase high performance liquid chromatography (rp-HPLC) and mass spectrometric (MS) analysis. A model peptide (Ac-QELHKLQ–NHCH₃) derived from salmon calcitonin was first labeled with a heterobifunctional cross-linker NHS-diazirine (succinimidyl 4,4′-azipentanoate; SDA) at Lys5 in solution, with ∼100% conversion. The SDA labeled peptide was then formulated with the following excipients at a 1:400 molar ratio and lyophilized: sucrose, trehalose, mannitol, histidine, arginine, urea, and NaCl. The lyophilized samples and corresponding solution controls were exposed to UV at 365 nm to induce photolytic labeling, and the products were identified by MS and quantified with rp-HPLC or MS. Peptide–excipient adducts were detected in the lyophilized solids except the NaCl formulation. With the exception of the histidine formulation, peptide–excipient adducts were not detected in solution and the fractional conversion to peptide–water adducts in solution was significantly greater than in lyophilized solids, as expected. In lyophilized solids, the fractional conversion to peptide–water adducts was poorly correlated with bulk moisture content, suggesting that the local water content near the labeled lysine residue differs from the measured bulk average. In lyophilized solids, the fractional conversion to peptide–excipient adducts was assessed using MS extracted ion chromatograms (EIC); subject to the assumption of equal ionization efficiencies, the fractional conversion to excipient adducts varied with excipient type. The results demonstrate that the local environment near the lysine residue of the peptide in the lyophilized solids can be quantitatively probed with a photolytic labeling method

Protein G, Protein A and Protein A-Derived Peptides Inhibit the Agitation Induced Aggregation of IgG

Controlling and preventing aggregation is critical to the development of safe and effective antibody drug products. The studies presented here test the hypothesis that protein A and protein G inhibit the agitation-induced aggregation of IgG. The hypothesis is motivated by the enhanced conformational stability of proteins upon ligand binding and the specific binding affinity of protein A and protein G to the Fc region of IgG. The aggregation of mixed human IgG from pooled human plasma was induced by agitation alone or in the presence of (i) protein A, (ii) protein G or (iii) a library of 24 peptides derived from the IgG-binding domain of protein A. Aggregation was assessed by UV spectroscopy, SDS–PAGE, high performance size-exclusion chromatography (HP-SEC), dynamic light scattering (DLS) and fluorescence spectroscopy. Additional information on IgG–ligand interactions was obtained using differential scanning fluorimetry (DSF) and competitive binding studies. The results demonstrate that protein A provides near-complete inhibition of agitation-induced aggregation, while protein G and two peptides from the peptide library show partial inhibition. The findings indicate that the IgG protein A-binding site is involved in the agitation-induced aggregation of IgG, and suggest a dominant role of colloidal interactions

Localized Hydration in Lyophilized Myoglobin by Hydrogen–Deuterium Exchange Mass Spectrometry. 2. Exchange Kinetics

Solid-state hydrogen–deuterium exchange with mass spectrometric analysis (ssHDX) is a promising method for characterizing proteins in amorphous solids. Though analysis of HDX kinetics is informative and well-established in solution, application of these methods to solid samples is complicated by possible heterogeneities in the solid. The studies reported here provide a detailed analysis of the kinetics of hydration and ssHDX for equine myoglobin (Mb) in solid matrices containing sucrose or mannitol. Water sorption was rapid relative to ssHDX, indicating that ssHDX kinetics was not limited by bulk water transport. Deuterium uptake in solids was well-characterized by a biexponential model; values for regression parameters provided insight into differences between the two solid matrices. Analysis of the widths of peptide mass envelopes revealed that, in solution, an apparent EX2 mechanism prevails, consistent with native conformation of the protein. In contrast, in mannitol-containing samples, a smaller non-native subpopulation exchanges by an EX1-like mechanism. Together, the results indicate that the analysis of ssHDX kinetic data and of the widths of peptide mass envelopes is useful in screening solid formulations of protein drugs for the presence of non-native species that cannot be detected by amide I FTIR

Localized Hydration in Lyophilized Myoglobin by Hydrogen–Deuterium Exchange Mass Spectrometry. 1. Exchange Mapping

The local effects of hydration on myoglobin (Mb) in solid matrices containing mannitol or sucrose (1:1 w/w, protein:additive) were mapped using hydrogen–deuterium exchange with mass spectrometric analysis (HDX–MS) at 5 °C and compared to solution controls. Solid powders were exposed to D₂O­(g) at controlled activity (<i>a</i>_w) followed by reconstitution and analysis of the intact protein and peptides produced by pepsin digestion. HDX varied with matrix type, <i>a</i>_w, and position along the protein backbone. HDX was less in sucrose matrices than in mannitol matrices at all <i>a</i>_w while the difference in solution was negligible. Differences in HDX in the two matrices were detectable despite similarities in their bulk water content. The extent of exchange in solids is proposed as a measure of the hydration of exchangeable amide groups, as well as protein conformation and dynamics; pepsin digestion allows these effects to be mapped with peptide-level resolution

Photolytic Cross-Linking to Probe Protein–Protein and Protein–Matrix Interactions in Lyophilized Powders

Protein structure and local environment in lyophilized formulations were probed using high-resolution solid-state photolytic cross-linking with mass spectrometric analysis (ssPC–MS). In order to characterize structure and microenvironment, protein–protein, protein–excipient, and protein–water interactions in lyophilized powders were identified. Myoglobin (Mb) was derivatized in solution with the heterobifunctional probe succinimidyl 4,4′-azipentanoate (SDA) and the structural integrity of the labeled protein (Mb-SDA) confirmed using CD spectroscopy and liquid chromatography/mass spectrometry (LC–MS). Mb-SDA was then formulated with and without excipients (raffinose, guanidine hydrochloride (Gdn HCl)) and lyophilized. The freeze-dried powder was irradiated with ultraviolet light at 365 nm for 30 min to produce cross-linked adducts that were analyzed at the intact protein level and after trypsin digestion. SDA-labeling produced Mb carrying up to five labels, as detected by LC–MS. Following lyophilization and irradiation, cross-linked peptide–peptide, peptide–water, and peptide–raffinose adducts were detected. The exposure of Mb side chains to the matrix was quantified based on the number of different peptide–peptide, peptide–water, and peptide–excipient adducts detected. In the absence of excipients, peptide–peptide adducts involving the CD, DE, and EF loops and helix H were common. In the raffinose formulation, peptide–peptide adducts were more distributed throughout the molecule. The Gdn HCl formulation showed more protein–protein and protein–water adducts than the other formulations, consistent with protein unfolding and increased matrix interactions. The results demonstrate that ssPC–MS can be used to distinguish excipient effects and characterize the local protein environment in lyophilized formulations with high resolution

A Cooperative Folding Unit as the Structural Link for Energetic Coupling within a Protein

Previously, we demonstrated that binding of a ligand to <i>Escherichia coli</i> cofactor-dependent phosphoglycerate mutase (dPGM), a homodimeric protein, is energetically coupled with dimerization. The equilibrium unfolding of dPGM occurs with a stable, monomeric intermediate. Binding of several nonsubstrate metabolites stabilizes the dimeric native form over the monomeric intermediate, reducing the population of the intermediate. Both the active site and the dimer interface appear to be unfolded in the intermediate. We hypothesized that a loop containing residues 118–152 was responsible for the energetic coupling between the dimer interface and the distal active site and was unfolded in the intermediate. Here, we investigated the structure of the dPGM intermediate by probing side-chain interactions and solvent accessibility of the peptide backbone. By comparing the effect of a mutation on the global stability and the stability of the intermediate, we determine an equilibrium φ value (φ_eq value), which provides information about whether side-chain interactions are retained or lost in the intermediate. Hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) was used to investigate differences in the solvent accessibility of the peptide backbone in the intermediate and native forms of dPGM. The results of φ_eq value analysis and HDX-MS reveal the least stable folding unit of dPGM, which is unfolded in the intermediate and links the active site to the dimer interface. The structure of the intermediate reveals how the cooperative network of residues in dPGM gives rise to the observed energetic coupling between dimerization and ligand binding

Probing the Conformation of an IgG1 Monoclonal Antibody in Lyophilized Solids Using Solid-State Hydrogen–Deuterium Exchange with Mass Spectrometric Analysis (ssHDX-MS)

Therapeutic proteins are often formulated as lyophilized products to improve their stability and prolong shelf life. The stability of proteins in the solid-state has been correlated with preservation of native higher order structure and/or molecular mobility in the solid matrix, with varying success. In the studies reported here, we used solid-state hydrogen–deuterium exchange with mass spectrometric analysis (ssHDX-MS) to study the conformation of an IgG1 monoclonal antibody (mAb) in lyophilized solids and related the extent of ssHDX to aggregation during storage in the solid phase. The results demonstrate that the extent of ssHDX correlated better with aggregation rate during storage than did solid-state Fourier-transform infrared (ssFTIR) spectroscopic measurements. Interestingly, adding histidine to sucrose at different formulation pH conditions decreased aggregation of the mAb, an effect that did not correlate with structural or conformational changes as measured by ssFTIR or ssHDX-MS. Moreover, peptide-level ssHDX-MS analysis in four selected formulations demonstrated global changes across the structure of the mAb when lyophilized with sucrose, trehalose, or mannitol, whereas site-specific changes were observed when lyophilized with histidine as the sole excipient

Solid-State Hydrogen–Deuterium Exchange Mass Spectrometry: Correlation of Deuterium Uptake and Long-Term Stability of Lyophilized Monoclonal Antibody Formulations

Solid state hydrogen–deuterium exchange with mass spectrometric analysis (ssHDX-MS) has been used to assess protein conformation and matrix interactions in lyophilized solids. ssHDX-MS metrics have been previously correlated to the formation of aggregates of lyophilized myoglobin on storage. Here, ssHDX-MS was applied to lyophilized monoclonal antibody (mAb) formulations and correlated to their long-term stability. After exposing lyophilized samples to D₂O­(g), the amount of deuterium incorporated at various time points was determined by mass spectrometry for four different lyophilized mAb formulations. Hydrogen–deuterium exchange data were then correlated with mAb aggregation and chemical degradation, which was obtained in stability studies of >2.5 years. Deuterium uptake on ssHDX-MS of four lyophilized mAb formulations determined at the initial time point prior to storage in the dry state was directly and strongly correlated with the extent of aggregation and chemical degradation during storage. Other measures of physical and chemical properties of the solids were weakly or poorly correlated with stability. The data demonstrate, for the first time, that ssHDX-MS results are highly correlated with the stability of lyophilized mAb formulations. The findings thus suggest that ssHDX-MS can be used as an early read-out of differences in long-term stability between formulations helping to accelerate formulation screening and selection