Solid-state Hydrogen Deuterium Exchange Research Articles

Probing the Protein-Excipient Interaction in the Orally Delivered Protein by Solid-State Hydrogen-Deuterium Exchange Mass Spectrometry and Molecular Dynamics.

The oral administration of protein therapeutics in solid dosage form is gaining popularity due to its benefits, such as improved medication adherence, convenience, and ease of use for patients compared to traditional parental delivery. However, formulating oral biologics presents challenges related to pH barriers, enzymatic breakdown, and poor bioavailability. Therefore, understanding the interaction between excipients and protein therapeutics in the solid state is crucial for formulation development. In this Letter, we present a case study focused on investigating the role of excipients in protein aggregation during the production of a solid dosage form of a single variable domain on a heavy chain (VHH) protein. We employed solid-state hydrogen-deuterium exchange coupled with mass spectrometry (ssHDX-MS) at both intact protein and peptide levels to assess differences in protein-excipient interactions between two formulations. ssHDX-MS analysis revealed that one formulation effectively prevents protein aggregation during compaction by blocking β-sheets across the VHH protein, thereby preventing β-sheet-β-sheet interactions. Spatial aggregation propensity (SAP) mapping and cosolvent simulation from molecular dynamics (MD) simulation further validated the protein-excipient interaction sites identified through ssHDX-MS. Additionally, the MD simulation demonstrated that the interaction between the VHH protein and excipients involves hydrophilic interactions and/or hydrogen bonding. This novel approach holds significant potential for understanding protein-excipient interactions in the solid state and can guide the formulation and process development of orally delivered protein dosage forms, ultimately enhancing their efficacy and stability.

Understanding the Impact of Protein-Excipient Interactions on Physical Stability of Spray-Dried Protein Solids.

Mannitol, leucine, and trehalose have been widely used in spray-dried formulations, especially for inhalation formulations. The individual contribution of these excipients on protein physical stability in spray-dried solids was studied here using bovine serum albumin (BSA) as a model protein. The spray-dried solids were characterized with scanning electron microscopy, powder X-ray diffraction, and solid-state Fourier-transform infrared spectroscopy to analyze particle morphology, crystallinity, and secondary structure change, respectively. Advanced solid-state characterizations were conducted with solid-state hydrogen-deuterium exchange (ssHDX) and solid-state nuclear magnetic resonance (ssNMR) to explore protein conformation and molecular interactions in the context of the system physical stability. Trehalose remained amorphous after spray-drying and was miscible with BSA, forming hydrogen bonds to maintain protein conformation, whereby this system showed the least monomer loss in the stability study. As indicated by ssNMR, both crystalline and amorphous forms of mannitol existed in the spray-dried BSA-mannitol solids, which explained its partial stabilizing effect on BSA. Leucine showed the strongest crystallization tendency after spray-drying and did not provide a stabilizing effect due to substantial immiscibility and phase separation with BSA as a result of crystal formation. This work showed novel applications of ssNMR in examining protein conformation and protein-excipient interaction in dry formulations. Overall, our results demonstrate the pivotal role of advanced solid-state characterization techniques in understanding the physical stability of spray-dried protein solids.

Pharmaceutical protein solids: Drying technology, solid-state characterization and stability.

Despite the boom in biologics over the past decade, the intrinsic instability of these large molecules poses significant challenges to formulation development. Almost half of all pharmaceutical protein products are formulated in the solid form to preserve protein native structure and extend product shelf-life. In this review, both traditional and emerging drying techniques for producing protein solids will be discussed. During the drying process, various stresses can impact the stability of protein solids. However, understanding the impact of stress on protein product quality can be challenging due to the lack of reliable characterization techniques for biological solids. Both conventional and advanced characterization techniques are discussed including differential scanning calorimetry (DSC), solid-state Fourier transform infrared spectrometry (ssFTIR), solid-state fluorescence spectrometry, solid-state hydrogen deuterium exchange (ssHDX), solid-state nuclear magnetic resonance (ssNMR) and solid-state photolytic labeling (ssPL). Advanced characterization tools may offer mechanistic investigations into local structural changes and interactions at higher resolutions. The continuous exploration of new drying techniques, as well as a better understanding of the effects caused by different drying techniques in solid state, would advance the formulation development of biological products with superior quality.

Stability of antibody drug conjugate formulations evaluated using solid-state hydrogen-deuterium exchange mass spectrometry.

Antibody drug conjugates (ADCs) have been at the forefront in cancer therapy due to their target specificity. All the FDA approved ADCs are developed in lyophilized form to minimize instability associated with the linker that connects the cytotoxic drug and the antibody during shipping and storage. We present here solid-state hydrogen-deuterium exchange with mass spectrometric analysis (ssHDX-MS) as a tool to analyze protein structure and matrix interactions for formulations of an ADC with and without commonly used excipients. We compared results of the ssHDX-MS with accelerated stability results using size-exclusion chromatography and determined that the former technique was able to successfully identify the destabilizing effects of mannitol and polysorbate 80. In comparison, Fourier-transform infrared spectroscopy results were inconclusive. The agreement between ssHDX-MS and stressed stability studies supports the potential of ssHDX-MS as a method of predicting relative stability of different formulations.

Effects of temperature and relative humidity in D2O on solid-state hydrogen deuterium exchange mass spectrometry (ssHDX-MS)

Lyophilized powders containing myoglobin and various excipients were subjected to ssHDX-MS at different temperatures and D2O vapor activity (RH). Deuterium incorporation was fitted to a bi-exponential association model for each formulation and the dependence of regression parameters on temperature and RH was evaluated. Data fitted best to a simplified model in which the slow exponential term was considered invariant with temperature and RH while the fast exponential term retained its temperature and RH dependence. This suggests that rapid rate processes such as water vapor sorption and initial deuterium labeling may be more dependent on temperature and RH than slower processes such as sequential exchange and transport in the solid matrix.

Effects of drying method and excipient on the structure and physical stability of protein solids: Freeze drying vs. spray freeze drying

This study aims to determine the impacts of drying method and excipient on changes in protein structure and physical stability of model protein solids. Protein solids containing one of two model proteins (lysozyme or myoglobin) were produced with or without excipients (sucrose or mannitol) using freeze drying or spray freeze drying (SFD). The protein powders were then characterized using solid-state Fourier transform infrared spectroscopy (ssFTIR), differential scanning calorimetry (DSC), circular dichroism spectrometry (CD), size exclusion chromatography (SEC), BET surface area measurements and solid-state hydrogen deuterium exchange with mass spectrometry (ssHDX-MS). ssFTIR and CD could identify little to no difference in structure of the proteins in the formulation. ssHDX-MS was able to identify the population heterogeneity, which was undetectable by conventional characterization techniques of ssFTIR and CD. ssHDX-MS metrics such as Dmax and peak area showed a good correlation with the protein physical instability (loss of the monomeric peak area by size exclusion chromatography) in 90-day stability studies conducted at 40 °C for lysozyme. Higher specific surface area was associated with greater loss in monomer content for myoglobin-mannitol formulations as compared to myoglobin-only formulations. Spray freeze drying seems a viable manufacturing technique for protein solids with appropriate optimization of formulations. The differences observed within the formulations and between the processes using ssHDX-MS, BET surface area measurements and SEC in this study provide an insight into the influence of drying methods and excipients on protein physical stability.

Prehydration and the Reversibility of Solid-State Hydrogen-Deuterium Exchange.

The reversibility of solid-state hydrogen-deuterium exchange (ssHDX) and the effects of prehydration on the rate and extent of deuterium incorporation were evaluated using poly-d,l-alanine (PDLA) peptides colyophilized with various excipients. In prehydration studies, samples were equilibrated at a controlled relative humidity (6% or 11% RH) for 12 h and then transferred to corresponding D2O humidity conditions (6% or 11% RD) for deuterium labeling. In amorphous samples, the rate and extent of deuterium incorporation were similar in prehydrated samples and controls not subjected to prehydration. In reversibility studies, PDLA samples were maximally deuterated in controlled D2O humidity conditions (6% or 11% RD) and then transferred to corresponding H2O relative humidity (0%, 6%, 11%, or 43% RH). Hysteresis in deuterium removal was observed when compared with the deuterium incorporation kinetics for all formulations and conditions, confirming that the reaction is reversible in the solid state and that the forward and reverse processes differ. The extent of deuterium loss reached a plateau that depended on the delabeling relative humidity. Reverse reaction rate constants were quantified using a first-order kinetic model, a limiting case of the reversible first-order model applicable under sink conditions. For other conditions, plateau (steady-state) deuteration levels were related to forward and reverse rate constants in a reversible first-order kinetic model. The results support a mechanistic interpretation of ssHDX kinetics as a reversible first-order process, in which the forward (deuteration) rate depends on the activity of the deuterium donor.

Effects of Secondary Structure on Solid-State Hydrogen-Deuterium Exchange in Model α-Helix and β-Sheet Peptides.

The effects of peptide secondary structure on the rate and extent of deuterium incorporation in solid-state hydrogen deuterium exchange mass spectrometry (ssHDX-MS) were assessed. Unstructured poly-d,l-alanine (PDLA) peptides, an α-helical model peptide (peptide A) and a β-sheet model peptide (peptide B), were co-lyophilized with various excipients. Peptide structures were confirmed in solution using circular dichroism (CD) spectroscopy and in the solid state with Fourier transform infrared (FTIR) spectroscopy. ssHDX-MS was conducted at two relative humidities (11 and 23% RH D2O) and deuterium uptake kinetics were monitored over 10 days. The relative contributions of peptide secondary structure and matrix interactions to deuteration incorporation were evaluated by comparing the ssHDX-MS kinetic data of peptide A and peptide B with PDLA of similar molecular weight. The results demonstrate that both peptide secondary structure and interactions with the solid matrix contribute to the protection from exchange in ssHDX-MS. A quantitative data analysis and interpretation method is presented, in which the number of protected amide bonds is calculated as the difference between the maximum deuterium incorporation in solution and in solid samples.

Optimizing the Formulation and Lyophilization Process for a Fragment Antigen Binding (Fab) Protein Using Solid-State Hydrogen-Deuterium Exchange Mass Spectrometry (ssHDX-MS).

Solid-state hydrogen-deuterium exchange with mass spectrometry (ssHDX-MS) was evaluated as an analytical method to rapidly screen and select an optimal lyophilized fragment antigen binding protein (Fab) formulation and the optimal lyophilization cycle. ssHDX-MS in lyophilized Fab formulations, varying in stabilizer type and stabilizer/protein ratio, was conducted under controlled humidity and temperature. The extent of deuterium incorporation was measured using mass spectrometry and correlated with solid-state stress degradation at 50 °C as measured by size exclusion chromatography (SEC) and ion-exchange chromatography (IEC). ssHDX-MS was also used to evaluate the impact of three different types of lyophilization processing on storage stability: controlled ice nucleation (CN), uncontrolled ice nucleation (UCN), and annealing (AN). The extent of deuterium incorporation for different Fab formulations agreed with the order of solid-state stress degradation, with formulations having lower deuterium incorporation showing lower stress-induced degradation (aggregation and charge modifications). For lyophilization processing, no significant effect of ice nucleation was observed in either solid-state stress degradation or in the extent of deuterium incorporation for high concentration Fab formulations (25 mg/mL). In contrast, for low concentration Fab formulations (2.5 mg/mL), solid-state stability from different lyophilization processes correlated with the extent of deuterium incorporation. The order of solid-state degradation (AN < CN < UCN) was the same as the extent of deuterium incorporation on ssHDX-MS (AN < CN < UCN). The extent of deuterium incorporation on ssHDX-MS correlated well with the solid-state stress degradation for different Fab formulations and lyophilization processing methods. Thus, ssHDX-MS can be used to rapidly screen and optimize the formulation and lyophilization process for a lyophilized Fab, reducing the need for time-consuming stress degradation studies.

Effects of ionic interactions on protein stability prediction using solid-state hydrogen deuterium exchange with mass spectrometry (ssHDX-MS).

Deuterium incorporation in solid-state hydrogen deuterium exchange with mass spectrometry (ssHDX-MS) has been correlated with protein aggregation on storage in sugar-based solid matrices. Here, the effects of sucrose, arginine and histidine buffer on the rate of aggregation of a lyophilized monoclonal antibody (mAb) were assessed using design of experiments (DoE) and response surface methodology. Lyophilized formulations were characterized using ssHDX-MS and Fourier transform infrared spectroscopy (ssFTIR) to assess potential correlation with stability in solid state. The samples were subjected to storage stability at 5 °C and stressed stability at 40 °C/75% RH for 6 months, and the aggregation rate was measured using size exclusion chromatography (SEC). Different levels of arginine had no significant effect on deuterium uptake in ssHDX-MS, although stability studies showed that aggregation rate decreased with increasing arginine concentration. Similarly, when histidine buffer was replaced with phosphate buffer at the same pH and molarity, ssHDX-MS showed no differences in deuterium uptake, but storage stability studies showed a significant increase in aggregation rate. The results suggest that proteins can be stabilized in amorphous solids by ionic interactions which ssHDX-MS does not detect, an important indication of the limitations of the method.

Solid-State Hydrogen-Deuterium Exchange Mass Spectrometry (ssHDX-MS) of Lyophilized Poly-d,l-Alanine.

Solid-state hydrogen-deuterium exchange mass spectrometry (ssHDX-MS) has been developed to study proteins in amorphous solids, but the relative contributions of protein structure and protein-matrix interactions to exchange are not known. In this work, short unstructured poly-d,l-alanine (PDLA) peptides were colyophilized with sucrose, trehalose, mannitol, sodium chloride, or guanidine hydrochloride to quantify the contributions of protein-matrix interactions to deuterium uptake in ssHDX-MS in the absence of a higher order structure. Deuterium incorporation differed with the excipient type and relative humidity (RH) in D2O(g), effects that were not observed in solution controls and are not described by the Linderstrom-Lang model for solution HDX. A reversible pseudo first-order kinetic model for deuterium uptake in ssHDX-MS is proposed. The model agrees with the experimentally observed dependences of the apparent deuteration rate and plateau value on RH in ssHDX-MS of PDLA and reduces to the Linderstrom-Lang limit when the forward rate of exchange is much greater than the reverse rate.

Use of MALDI-MS with solid-state hydrogen deuterium exchange for semi-automated assessment of peptide and protein physical stability in lyophilized solids

Biological therapeutics are established as major contributors to the pharmaceutical pipeline. Many of these biological drugs are lyophilized to preserve their conformation and reduce decomposition during storage and shipping. Therefore, understanding and controlling the effects of lyophilization on protein higher order structure is critical for commercialization of biologics. Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS) is a well-established technique for studying protein higher order structure. Previous publications have demonstrated a solid state HDX (ssHDX) method for labeling formulated, lyophilized proteins to assess their physical stability during, but this process still suffered from low throughput and undesired back exchange. Recently, our group described a method combining HDX-MS with MALDI to greatly reduce the time of analysis and nearly eliminate H/D back-exchange, but that method was not suited for interrogating solid samples. This work integrates the two techniques to assess and predict the stability of peptides and proteins following mixing and lyophilization with various excipient formulations. Sample mixing and handling were performed through the use of a bench-top robotics and programmed data MALDI-MS acquisition allowed for monitoring deuterium incorporation for dried peptides and protein samples following continuous labeling with D2O vapor. Effects of excipients upon peptide stability were also tracked and compared to a control for a three day labeling time course. This workflow is automated and free from back-exchange. As demonstrated by deuterium retention of bradykinin, these features serve to reduce experimental error normally associated with conventional deuterium exchange experiments. The proposed union of MALDI-MS and ssHDX can be applied to study higher order structure of proteins and peptides and the effects of added excipients in an environment that closely resembles the storage and shipping conditions of biopharmaceuticals and may be beneficial in giving insights studying protein structural dynamics in solids.

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.

Effects of Drying Process on an IgG1 Monoclonal Antibody Using Solid-State Hydrogen Deuterium Exchange with Mass Spectrometric Analysis (ssHDX-MS).

Lyophilization and spray drying are widely used to manufacture solid forms of therapeutic proteins. Lyophilization is used to stabilize proteins vulnerable to degradation in solution, whereas spray drying is mainly used to prepare inhalation powders or as an alternative to freezing for storing bulk drug substance. Both processes impose stresses that may adversely affect protein structure, stability and bioactivity. Here, we compared lyophilization with and without controlled ice nucleation, and spray drying for their effects on the solid-state conformation and matrix interactions of a model IgG1 monoclonal antibody (mAb). Solid-state conformation and matrix interactions of the mAb were probed using solid-state hydrogen-deuterium exchange with mass spectrometric analysis (ssHDX-MS), and solid-state Fourier transform infrared (ssFTIR) and solid-state fluorescence spectroscopies. mAb conformation and/or matrix interactions were most perturbed in mannitol-containing samples and the distribution of states was more heterogeneous in sucrose and trehalose samples that were spray dried. The findings demonstrate the sensitivity of ssHDX-MS to changes weakly indicated by spectroscopic methods, and support the broader use of ssHDX-MS to probe formulation and process effects on proteins in solid samples.

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.

Process and Formulation Effects on Protein Structure in Lyophilized Solids Using Mass Spectrometric Methods

Myoglobin (Mb) was lyophilized in the absence (Mb-A) and presence (Mb-B) of sucrose in a pilot-scale lyophilizer with or without controlled ice nucleation. Cake morphology was characterized using scanning electron microscopy, and changes in protein structure were monitored using solid-state Fourier-transform infrared spectroscopy, solid-state hydrogen-deuterium exchange–mass spectrometry, and solid-state photolytic labeling–mass spectrometry (ssPL-MS). The results showed greater variability in nucleation temperature and irregular cake structure for formulations lyophilized without controlled nucleation. Controlled nucleation resulted in nucleation at ∼(−5°C) and uniform cake structure. Formulations containing sucrose showed better retention of protein structure by all measures than formulations without sucrose. Samples lyophilized with and without controlled nucleation were similar by most measures of protein structure. However, ssPL-MS showed the greatest photoleucine incorporation and more labeled regions for Mb-B lyophilized with controlled nucleation. The data support the use of solid-state hydrogen-deuterium exchange–mass spectrometry and ssPL-MS to study formulation and process-induced conformational changes in lyophilized proteins.

Characterizing Protein Structure, Dynamics and Conformation in Lyophilized Solids.

The long-term stability of protein therapeutics in the solid-state depends on the preservation of native structure during lyophilization and in the lyophilized powder. Proteins can reversibly or irreversibly unfold upon lyophilization, acquiring conformations susceptible to degradation during storage. Therefore, characterizing proteins in the dried state is crucial for the design of safe and efficacious formulations. This review summarizes the basic principles and applications of the analytical techniques that are commonly used to characterize protein structure, dynamics and conformation in lyophilized solids. The review also discusses the applications of recently developed mass spectrometry based methods (solid-state hydrogen deuterium exchange mass spectrometry (ssHDX-MS) and solid-state photolytic labeling mass spectrometry (ssPL-MS)) and their ability to study proteins in the solid-state at high resolution.

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.