A c*-guided high-temperature rheological approach and solid-state NMR (ssNMR) 1H T1ρ and 1H T1 relaxation time measurements were used to estimate the miscibility of celecoxib (CEL)/polyvinylpyrrolidone (PVP) amorphous solid dispersions (ASDs) prepared by melt quench and spray drying. The ssNMR spin diffusion method is capable of investigating the miscibility of ASDs processed by the two methods, as it measures as-is ASD powders, while the c*-guided high-temperature rheological approach cannot, since it erases the processing history. Our results indicate a general agreement of the estimated miscibility of ASDs prepared by the two processing methods. The crystallization tendency is significantly different when the polymer concentration c is below and above c*. When c < c*, the onset of crystallization in dilute CEL/PVP ASDs is approximately identical to that of neat amorphous CEL. However, when c > c*, the first evidence of CEL crystallization is significantly delayed, indicating a reduced crystallization propensity. These results confirm that the efficient and material-sparing rheological approach can be used to estimate the miscibility (limit) and predict stability against crystallization for both melt-extruded and spray-dried ASDs. Our findings are useful in the rational and efficient design of robust ASDs with a desirable stability against crystallization during long-term storage.

Spray drying is an emerging continuous manufacturing approach with significant potential for producing stable solid formulations of biologic drug products such as peptides, proteins and oligonucleotides. However, these sensitive molecules on exposure to thermal, shear, and interfacial stresses during spray drying may experience structural perturbations that compromise long-term stability. This study evaluated several polymeric excipients as stabilizers in spray-dried monoclonal antibody formulations and compared their performance with conventional sugar excipients, trehalose and mannitol. Formulations were prepared at a 1:1 (w/w) protein-to-excipient ratio and spray dried, and the resulting powders were subjected to accelerated storage at 40 °C. Particle size distribution, morphology, and residual moisture content were characterized. Protein physical stability and secondary structure were evaluated using size exclusion chromatography and solid-state Fourier transform infrared spectroscopy, respectively. Solid-state nuclear magnetic resonance spectroscopy and X-ray photoelectron spectroscopy were used to study protein-excipient phase miscibility and particle surface composition, respectively, in the spray-dried solids. Hydrolyzed gelatin provided the most robust stabilization of the mAb, resulting in a fivefold reduction in monomer loss compared to trehalose after 90 days storage at 40 °C. (2-Hydroxypropyl)-β-cyclodextrin showed stabilization comparable to trehalose while reducing the mAb surface exposure. High molecular weight polyvinylpyrrolidone (PVP K90) showed better mAb stabilization compared to its low molecular weight grade (PVP K30). Whereas dextran, sodium carboxymethyl cellulose, and hydroxypropyl methylcellulose were less effective in their ability to stabilize the mAb. These findings highlight the critical role of excipient selection in improving the stability of spray-dried monoclonal antibody formulations.

Nitrosamines were first identified in the 1950s as carcinogenic contaminants in food, tobacco, and industrial chemicals. Among them, N-nitrosodimethylamine (NDMA) is particularly dangerous due to its high carcinogenicity and frequent occurrence in drugs, such as ranitidine hydrochloride. Recent studies indicate that NDMA formation in solid-state ranitidine hydrochloride is driven by solid-state reactive species (SSRS), such as crystal defects, amorphous regions, and other high-energy sites within the drug crystal. In this paper, we demonstrate that NDMA formation in ranitidine hydrochloride can be essentially stopped by reducing the presence of SSRS in the drug substance through recrystallization. Well-controlled recrystallized ranitidine hydrochloride that resulted in high-quality crystals exhibited significantly lower NDMA formation compared to as-received material when stored at 60 °C and <2% relative humidity. Cryoground samples showed substantially increased reactivity, whereas subsequent well-controlled recrystallization restored stability, confirming the critical role of crystal quality. In contrast, poorly controlled recrystallization produced low-quality crystals with elevated SSRS, leading to higher degradation rates. These results eventually motivated us to test the role of oxygen in the degradation process for ranitidine hydrochloride. Cryoground ranitidine hydrochloride samples exhibited minimal reactivity under vacuum or nitrogen, elevated reactivity in air (∼21% oxygen), and the highest nitrosamine formation in an oxygen-rich environment. These results suggest that well-controlled crystallization that produced high-quality crystals may be an extremely effective method to inhibit nitrosamine formation, as SSRS are needed to promote reactivity, and could be an alternative and/or complementary approach to the current strategy of adding antioxidants and/or pH modification to formulations to minimize nitrosamine formation.

Abstract Trehalose is a commonly used lyoprotectant for stabilizing proteins in lyophilized formulations. Its physical state plays a critical role in maintaining both the physical and chemical stability of proteins. In this study, three types of protein/trehalose systems were studied to understand the impact of trehalose crystallization on protein aggregation in the lyophilized state. The three systems included BSA60, which contains approximately 60% monomers of bovine serum albumin (BSA) and 40% aggregates; BSA95, with about 95% monomers and 5% aggregates; and a monoclonal antibody (mAb), which had little aggregation (< 1%). Each was formulated with trehalose and lyophilized using varying annealing durations. Trehalose crystallized after 24 h of annealing in BSA60 formulations, whereas BSA95 and mAb formulations required 72 h of annealing to induce crystallization. Although crystalline trehalose was present in the 24-h annealed BSA60 samples after lyophilization, no additional aggregation was observed on storage. In contrast, BSA95 and mAb formulations, where crystalline trehalose was also observed after lyophilization, showed monomer loss at all time points (1 month–12 months) during storage at 50 °C. Overall, these results indicate that trehalose crystallization does not usually occur under typical lyophilization conditions, and generally requires prolonged annealing and a high trehalose-to-protein ratio. When crystallization does occur, it results in inhomogeneity, as shown by solid-state NMR spectroscopy, leading to reduced stability of the protein in the matrix.

Abstract Lyophilization is widely used to stabilize and enhance the shelf-life of protein drug products, including monoclonal antibodies (mAbs), bi- and tri-specific antibodies, antibody fragments, enzymes, etc. Disaccharides such as trehalose and sucrose are commonly used as stabilizers in lyophilized protein formulations. For these disaccharide excipients to be good stabilizers, it requires formation of rigid, immobile matrix where proteins are homogenously distributed. In this study, we investigated the relationship between molecular mobility, homogeneity, and long-term stability in lyophilized formulations of a mAb with trehalose or sucrose as stabilizers. We used solid-state nuclear magnetic resonance spectroscopy (ssNMR) to measure 1H T₁ and 1H T₁ρ relaxation times, probing the mobility and homogeneity of the components at 20–50 nm and 2–5 nm scales, respectively. Formulations with higher disaccharide content showed longer 1H T₁ relaxation times and greater monomer retention, consistent with a more rigid glassy matrix and enhanced stability. Relaxation times of mAb and disaccharide were closely matched in most formulations, indicating good homogeneity, which was essential for stability. However, exceptions were observed in samples where disaccharide crystallization occurred, leading to phase separation and inhomogeneity in the lyophilized matrix. Despite having long 1H T₁ times, these samples showed reduced stability, underscoring the importance of maintaining both low mobility and homogeneous amorphous structure. Overall, this study demonstrates that ssNMR relaxation measurements are effective predictors of lyophilized protein stability as they provides information on both matrix rigidity and component homogeneity in the solid state.

Spray-freeze drying (SFD) is a promising drying technique for stabilizing RNA lipid nanoparticles (RNA-LNPs) by converting them into solid-state formulations. This study examined the impact of the SFD process and the widely utilized stabilizing disaccharides, such as sucrose and trehalose, on the major properties (e.g. particle size, encapsulation efficiency) of RNA-LNPs post drying. Various process parameters, such as atomization, freezing, and drying temperature, were systematically evaluated, along with the impact of incorporating annealing post freezing step. Surface area measurements, solid-state particle size analysis, scanning electron microscopy, powder X-ray diffraction and solid-state NMR spectroscopy were employed to gain deeper insights into powder characteristics and matrix mobility. Our findings demonstrated that sucrose provided superior stabilization as compared to trehalose in our tested formulations and processes. Moreover, incorporation of the annealing process enhanced LNP stability. Results from the stability study revealed that the annealed 10

Protein-based therapeutics at high-concentrations often face significant development challenges, including high viscosity, limited solubility, poor injectability, and instability concerns. Non-aqueous protein suspensions offer a promising strategy to achieve high protein concentrations while maintaining acceptable viscosity and injectability. In this study, we evaluated the impact of two polymeric excipients (hydrolyzed gelatin, hydroxypropyl-β-cyclodextrin or HPβCD) and their combinations on the viscosity, injectability, and stability of high-concentration non-aqueous suspensions containing bovine serum albumin (BSA). Suspension viscosity was characterized by rheological measurement, while injectability was assessed using a custom-built setup to measure plunger force through a syringe with a 27G needle. Spray-dried powders and the corresponding suspensions were subjected to accelerated physical stability (monomer loss) studies. Protein stability and structural integrity were evaluated using size-exclusion chromatography (SEC), circular dichroism (CD), and solid-state NMR (ssNMR), while X-ray photoelectron spectroscopy (XPS) was used to assess surface chemical properties of spray-dried particles. Hydrolyzed gelatin provided approximately five-fold greater preservation of monomer content during storage, indicating enhanced protein stability (∼1.5% monomer loss in 90-day stressed storage). In contrast, HPβCD significantly improved injectability, reducing injection force by approximately 5 N compared with formulations containing protein alone (13 N reduced to 8 N). The combination of hydrolyzed gelatin and HPβCD yielded stable and injectable suspensions with protein loadings of 150 - 250 mg/mL. The current work highlighted the potential of polymer-based excipient systems for developing high-concentration injectable suspensions of proteins.

Pharmaceutical analysis is essential to drug development and quality assurance, ensuring that products meet stringent safety and efficacy standards. Quantitative solid-state NMR (qSSNMR) has become a key technique, enabling precise quantification and characterization of solid drug formulations. This mini-review highlights the evolution of qSSNMR, focusing on improvements in detection limits, resolution, and high-throughput capabilities. This review explores technical advancements and applications for analyzing complex pharmaceutical mixtures. While challenges remain for widespread adoption, efforts in automation, user-friendly software, and collaboration aim to address these.

The physical stability of two structurally similar drugs, indomethacin (IND) and indomethacin methyl ester (INDME), was investigated by comparison of thermodynamic, kinetic, and structural factors affecting crystallization. The impact of drug loading, storage temperature relative to the glass transition temperature (Tg), and hydrogen bonding ability has been explored for their relative importance as the cause for crystallization in amorphous solid dispersions (ASDs). IND or INDME and polyvinylpyrrolidone (PVP) K12 ASDs were formulated at varying drug loadings via cryomilling and melt-quenching. Differential scanning calorimetry was used to assign storage temperatures in the supercooled liquid and glassy states based on the Tg of each dispersion. The crystallization onset time (tc) was monitored using powder X-ray diffraction while Fourier-transform infrared spectroscopy monitored changes in hydrogen bonding. IND formed strong homogeneous and IND-PVP hydrogen bonds while there was scarce evidence of INDME hydrogen bonding. The tc of IND and INDME ASDs was inversely related to drug loading and storage temperature. However, systems with 80% IND or greater exhibited a deviation from the exponential relationship in tc as Tg was approached. At high drug loadings, IND crystallized faster at temperatures near and slightly below Tg than at temperatures above Tg. IND has a greater thermodynamic driving force for crystallization relative to INDME at all temperatures above Tg. However, this is offset by a reduction in molecular mobility due to extensive hydrogen bonding with PVP. Near Tg, IND still has extensive translational mobility driven by the formation of IND-IND dimers during nucleation and crystallization and is proposed as a possible cause of the increased IND crystallization kinetics. Increased drug loading and temperature provide a larger thermodynamic driving force for crystallization and reduce the crystallization onset time. However, their relative contributions may change with decreasing temperature to the point where crystallization onset time above Tg may not be extrapolated below Tg. For the first time, diffusionless crystallization is observed in systems above 2% polymer to which a difference in thermodynamics, driven by hydrogen bond formation, is thought to be the cause. A better understanding of the causes of destabilization and their relative significance toward causing crystallization will help to make better informed decisions during formulation and storage.

There is a growing interest in developing solid formulations for protein products due to enhanced physico-chemical stability. Spray drying is a widely used process for the manufacturing of solid-state protein therapeutics for pulmonary delivery. Stabilizers such as trehalose are commonly used and play a critical role in preventing protein degradation during the process of spray drying and upon storage. However, the hygroscopicity of spray-dried trehalose can result in moisture absorption, leading to crystallization and phase separation, and consequently impacting protein stability. Amino acids such as L-leucine have been studied as excipients that protect the hygroscopic spray-dried formulations against high humidity conditions. However, the interactions of L-leucine with protein and trehalose are less understood. This study systematically examined the role of L-leucine and trehalose on the physical and aerosol stabilities of the spray-dried protein formulations. Our findings revealed that a L-leucine ratio of 46.7% w/w was adequate to inhibit the crystallization of amorphous trehalose and prevent particle agglomeration even under humidity conditions such as 75% RH, without significantly impacting protein stability.

The formation of nitrosamines, a class of potent carcinogen impurities, in pharmaceutical products such as ranitidine hydrochloride (RAN) has raised significant public health concerns and led to widespread product recalls. Previous studies have shown that RAN can degrade to N-nitrosodimethylamine (NDMA) after storage at 60 °C for several days. These studies also suggested a link between the degradation rate and the crystal morphology of the RAN. This study shows that NDMA formation in RAN is primarily driven by solid-state reactive species (SSRS) introduced during pharmaceutical manufacturing processes such as crystallization, milling, and grinding. Using cryogenic milling to introduce SSRS systematically, we assessed the stability of RAN at 60 °C and 0 % RH. Our results show a clear relationship between the amount of SSRS in RAN and the amount of NDMA formed upon stability, with cryoground samples exhibiting degradation rates up to two orders of magnitude higher than unprocessed samples. Furthermore, RAN tablets prepared with increased SSRS also showed accelerated NDMA formation. This study demonstrates the importance of mitigating SSRS in pharmaceutical manufacturing by mitigating the presence of SSRS in drug substances/products.

Stabilization of proteins by disaccharides in lyophilized formulations depends on the interactions between the protein and the disaccharide (system homogeneity) and the sufficiently low mobility of the system. Human serum albumin (HSA) was lyophilized with disaccharides (sucrose and/or trehalose) in different relative concentrations. Solid-state nuclear magnetic resonance (ssNMR) spectroscopy 1H T1 and 1H T1ρ relaxation times were measured to determine the homogeneity of the lyophilized systems on 20–50 and 1–3 nm domains, respectively, with 1H T1 relaxation times also being used to determine the β-relaxation rate. HSA/sucrose systems had longer 1H T1 relaxation times and were slightly more stable than HSA/trehalose systems in almost all cases shown. HSA/sucrose/trehalose systems have 1H T1 relaxation times between the HSA/sucrose and HSA/trehalose systems and did not result in a more stable system compared with binary systems. Inhomogeneity was evident in a sample containing relative concentrations of 10% HSA and 90% trehalose, suggesting trehalose crystallization during lyophilization. Under these stability conditions and with these ssNMR acquisition parameters, a 1H T1 relaxation time below 1.5 s correlated with an unstable sample, regardless of the disaccharide(s) used.

The utility of employing solid-state NMR (SSNMR) to assess parameters governing the stability of a lyophilized IgG2 protein was the focus of the present work. Specifically, the interaction between the sugar stabilizer (sucrose) and protein component was measured using SSNMR and compared to physical and chemical stability data obtained from thermally stressed samples. 1H T1 and 1H T1⍴ relaxation times were measured by SSMNR for 5 different formulation conditions, and the resultant values were used to examine local mobility and phase separation, respectively. From the SSNMR measurements, it was found local mobility decreased as the sucrose to protein weight ratio increased. The decrease in local mobility corresponded to an increase in storage stability (both chemical and physical) of the lyophilized solids up to a critical weight ratio of sucrose to protein. Additionally, 1H T1⍴ measurements obtained on formulations having higher protein to sucrose weight ratios indicated phase separation of the protein and sucrose phases was occurring, at least on a small scale. Along with an increase in local mobility, phase separation in these specific formulations is thought to have played a role in their decreased storage stability in the solid state.

In a lyophilized protein/disaccharide system, the ability of the disaccharide to form a homogeneous mixture with the protein and to slow the protein mobility dictates the stabilization potential of the formulation. Human serum albumin was lyophilized with sucrose or trehalose in histidine, phosphate, or citrate buffer. 1H T1 relaxation times were measured by solid-state NMR spectroscopy and were used to assess the homogeneity and mobility of the samples after zero, six, and twelve months at different temperatures. The mobility of the samples decreased after 6 and 12 months storage at elevated temperatures, consistent with structural relaxation of the amorphous disaccharide matrix. Formulations with sucrose had lower mobility and greater stability than formulations with trehalose.

Miscibility is critical in the prediction of stability against crystallization of amorphous solid dispersions (ASDs) in the solid state. However, currently available approaches for its determination are limited by both theoretical and practical considerations. Recently, a rheological approach guided by the polymer overlap concentration (c*) has been proposed for miscibility quantification of ASDs [J. Pharm. Sci., 112 (2023) 204−212] and shown to be useful in predicting both accelerated and long term physical stability without the presence of moisture. However, this approach can only be performed at high temperatures (slightly above the melting temperature, Tm, of drugs), and little is known about the difference in miscibility between high and low temperatures (e.g., below the glass transition temperature, Tg). Here we compare the miscibility of nifedipine (NIF)/polyvinylpyrrolidone (PVP) ASDs as determined by the rheological approach at 175°C (∼3°C above Tm of NIF) and solid state NMR (ssNMR) 1H T1 and T1ρ relaxation times at -20°C (∼66°C below Tg of NIF). Our results indicate an agreement between the two methods. For low molecular weight (Mw) PVP, T1ρ measurements are more consistent with the rheological approach, while T1 measurements are closer for relatively high Mw PVP. Our findings support the use of the c* based rheological approach for inferring miscibility of deeply cooled ASDs.

Changes in the protonation state of lyophilized proteins can impact structural integrity, chemical stability, and propensity to aggregate upon reconstitution. When a buffer is chosen, the freezing/drying process may result in dramatic changes in the protonation state of the protein due to ionization shift of the buffer. In order to determine whether protonation shifts are occurring, ionizable probes can be added to the formulation. Optical probes (dyes) have shown dramatic ionization changes in lyophilized products, but it is unclear whether the pH indicator is uniform throughout the matrix and whether the change in the pH indicator actually mirrors drug ionization changes. In solid-state NMR (SSNMR) spectroscopy, the chemical shift of the carbonyl carbon in carboxylic acids is very sensitive to the ionization state of the acid. Therefore, SSNMR can be used to measure ionization changes in a lyophilized matrix by employing a small quantity of an isotopically-labeled carboxylic acid species in the formulation. This paper compares the apparent pH of six trehalose-containing lyophilized buffer systems using SSNMR and UV-Vis diffuse reflectance spectroscopy (UVDRS). Both SSNMR and UVDRS results using two different ionization probes (butyric acid and bromocresol purple, respectively) showed little change in apparent acidity compared to the pre-lyophilized solution in a sodium citrate buffer, but a greater change was observed in potassium phosphate, sodium phosphate, and histidine buffers. While the trends between the two methods were similar, there were differences in the numerical values of equivalent pH (pHeq) observed between the two methods. The potential causes contributing to the differences are discussed.

Stability of the majority of pharmaceuticals and biopharmaceuticals depends on acidity/basicity of the environment. In aqueous solutions, acidity/basicity is commonly expressed using proton activity scale, pH, while definition and experimental measurements of acid-base relationships in frozen and freeze-dried materials are less straightforward. The chapter starts with a brief summary of the current understanding of several critical aspects of pH and apparent acidity/basicity as related to freezing and freeze-drying, whereas the main part of the chapter is focused on areas which are underrepresented in the pharmaceutical literature, with both overview of the literature and previously unpublished data presented. In particular, the following topics are covered: (i) Hammett acidity function and pH-equivalent (pHeq); (ii) Factors which impact apparent solid-state acidity in lyophiles; (iii) Solid-state acidity and chemical instability of lyophiles; (iv) Freezing fundamentals: quasi-liquid layer, polarity of the freeze-concentrated solution, and the Workman-Reynolds potential. Potential directions for future studies in this field are also outlined.

Pulmonary delivery of protein-based therapeutics, including antibodies, is a promising option for treating respiratory diseases. Spray drying is a widely used method for producing dry powder formulations with mannitol being a commonly used excipient for these inhalation formulations. There is limited research available concerning the utilization of mannitol as an excipient in the spray drying of proteins and its impact on aerosol performance. This study highlights the importance to understand mannitol’s potential role and impact in this context. To investigate the impact of mannitol on physical stability and aerosolization of spray-dried protein formulations, bovine serum albumin (BSA) was employed as a model protein and formulated with different concentrations of mannitol via spray drying. The spray-dried solids were characterized for their particle size using Malvern mastersizer and aerodynamic particle size using next generation impactor (NGI). Additionally, the solids were characterized with solid-state Fourier-transform infrared spectroscopy (ssFTIR), powder X-ray diffraction (PXRD), scanning electron microscopy (SEM) and solid-state nuclear magnetic resonance spectroscopy (ssNMR) to analyze the change in their secondary structure, crystallinity, particle morphology, and protein-excipient interaction, respectively. Size exclusion chromatography (SEC) was used to investigate changes in monomer content resulting from storage under stressed condition of 40 °C. Protein formulations containing more than 33 % mannitol by weight showed crystallization tendencies, causing an increase in monomer loss over time. ssNMR data also showed mixing heterogeneity of BSA and mannitol in the formulations with high mannitol contents. Futhermore, fine particle fraction (FPF) was found to decrease over time for the formulations containing BSA: Mannitol in the ratios of 2:1, 1:2, and 1:5, due to particle agglomeration induced by crystallization of mannitol. This study underscores the significant influence of excipients such as mannitol on the aerosol performance and storage stability of spray-dried protein formulations.

Solid-state nuclear magnetic resonance (NMR) spectroscopy (SSNMR) is a powerful analytical technique that can be utilized to study lyophilized formulations. This chapter details basics of SSNMR spectroscopy and ways that SSNMR can be utilized to assess important properties of lyophilized formulations including excipient solid form, excipient and drug interactions in the solid state, changes in ionization of a formulation component, and mobility in the lyophilized solid. A case study is presented that utilizes SSNMR to look at the interaction of sucrose, trehalose, and mannitol with the model proteins lysozyme and bovine serum albumin.

Background:Globally, approximately 800 women and 6400 newborns die around the time of childbirth each day. Many of these deaths could be prevented with high-quality emergency obstetric and newborn care (EmONC). The Monitoring Emergency Obstetric Care: A handbook guides strengthening EmONC services. However, the handbook contains limited quality of care measures. Our study identified and prioritised quality of care indicators for potential inclusion in the handbook, which is undergoing revision. Methods:We conducted a consultative scoping review, mapping, and prioritisation exercise to select a short list of indicators on facility-based maternal and newborn quality of care. Indicators were identified from literature searches and expert suggestions and organised by the categories of structure, process, and outcomes as defined in the World Health Organization's Standards for Improving Quality of Maternal and Newborn Care in Health Facilities. We focused on process indicators, encompassing the provision of care and experience of care during the intrapartum period, and developed a priority list of indicators using the selection criteria of relevance and feasibility. Experience of care indicators were also mapped against the Person-Centered Maternity Care (PCMC) scale. Results:We extracted a total of 3023 quality of care indicators. After removing out-of-scope and duplicate indicators and applying our selection criteria, we identified 20 provision of care indicators for possible inclusion in the revised EmONC handbook. We recommend including a score for experience of care that could be measured with the 30-item or the 13-item PCMC scale. We also identified 29 experience of care items not covered by the PCMC scale that could be used. Provider experience, patient safety, and quality of abortion care were identified as areas for which no or few indicators were found through our scoping review. Conclusions:Through a rigorous, consultative, and multi-step process, we selected a short list of process-related, facility-based quality of care indicators for emergency obstetric and newborn care. This list could be included in the EmONC handbook or used for other monitoring purposes. Country consultations to assess the utility and feasibility of the proposed indicators and their adaptation to local contexts will support their refinement and uptake. Registration:https://osf.io/msxbd (Open Science Framework).