Mechanism of Insoluble Aggregate Formation in a Reconstituted Solution of Spray-Dried Protein Powder.

BACKGROUND: Spray-drying is considered a promising alternative drying method to lyophilization (freeze-drying) for therapeutic proteins. Particle counts in reconstituted solutions of dried solid dosage forms of biologic drug products are closely monitored to ensure product quality. We found that high levels of particles formed after reconstitution of protein powders that had been spray-dried under suboptimal conditions. METHODS: Visible and subvisible particles were evaluated. Soluble proteins in solution before spray-drying and in the reconstituted solution of spray-dried powder were analyzed for their monomer content levels and melting temperatures. Insoluble particles were collected and analyzed by Fourier transform infrared microscopy (FTIR), and further analyzed with hydrogen-deuterium exchange (HDX). RESULTS: Particles observed after reconstitution were shown not to be undissolved excipients. FTIR confirmed their identity as proteinaceous in nature. These particles were therefore considered to be insoluble protein aggregates, and HDX was applied to investigate the mechanism underlying aggregate formation. Heavy-chain complementarity-determining region 1 (CDR-1) in the aggregates showed significant protection by HDX, suggesting CDR-1 was critical for aggregate formation. In contrast, various regions became more conformationally dynamic globally, suggesting the aggregates have lost protein structural integrity and partially unfolded after spray-drying. DISCUSSION: The spray-drying process could have disrupted the higher-order structure of proteins and exposed the hydrophobic residues in CDR-1 of the heavy chain, contributing to the formation of aggregate through hydrophobic interactions upon reconstitution of spray-dried powder. These results can contribute to efforts to design spray-dry resilient protein constructs and improve the robustness of the spray-drying process.

Effect of 'pH' on the Rate of Pyroglutamate Formation in Solution and Lyophilized Solids.

N-terminal glutamate can cyclize to form pyroglutamate (pGlu) in pharmaceutically relevant peptides and proteins. The reaction occurs nonenzymatically during storage for monoclonal antibodies and shows a strong 'pH' dependence in solution, but the solid-state reaction has not been studied in detail. This work investigates the effect of 'pH' and buffer species on pGlu formation for a model peptide (EVQLVESGGGLVQPGGSLR) in lyophilized solids and in solution. The model peptide was formulated from 'pH' 4 to 'pH' 9 in citrate, citrate-phosphate, phosphate, and carbonate buffers and stored at 50 °C for at least 10 weeks. pGlu formation and loss of the parent peptide were monitored by reversed-phase high-performance liquid chromatography. The apparent 'pH' dependence of the reaction rate in the solid state differed markedly from that in solution. Interestingly, in the 'pH' range often used to formulate mAbs ('pH' 5.5-6), the rate of pGlu formation in the solid state was greater than that in solution. The results have implications for the rational design of stable formulations of peptides and proteins, and for the transition from solid to solution formulations during development.

Percutaneous CT-Guided Cryoablation of the Celiac Plexus: A Retrospective Cohort Comparison with Ethanol.

PURPOSE: To retrospectively analyze and compare the incidence of diarrhea in patients who underwent cryoablation of the celiac plexus for intractable abdominal pain versus ethanol therapy over a 5-year period. MATERIALS AND METHODS: From June 2014 to August 2019, 83 patients were identified who underwent neurolysis of the celiac plexus for management of intractable abdominal pain by using either cryoablation (n = 39 [59% female; age range, 36-79 years old [average, 60 ± 11 years old]) or alcohol (n = 44 [48% female; age range, 29-76 years old [average, 60 ± 12 years old]). Pain scores and reports of procedure-related complications or side effects, with special attention to diarrhea and/or other gastrointestinal symptoms, were collected from follow-up visits at 1 week, 1 month, and 3 months post-intervention and were compared between groups. RESULTS: The mean time of follow-up was 17.7 days. Four patients who underwent cryoablation developed gastrointestinal symptoms consisting of 2 cases of nausea and vomiting and 2 cases of diarrhea (5.1%). Twelve patients who underwent ethanol ablation developed gastrointestinal symptoms, including 1 case of nausea, 3 cases of vomiting, and 9 cases of diarrhea (20.5%). There was a significantly higher incidence of both diarrhea (chi-squared likelihood ratio, P = .03) and overall gastrointestinal symptoms (chi-squared likelihood ratio, P = .04) in the ethanol group than in the cryoablation group. CONCLUSIONS: Cryoablation of the celiac plexus may provide a new treatment option for intractable abdominal pain, and it appears to have a lower incidence of diarrhea and fewer gastrointestinal side effects than ablation using ethanol.

Application of Magnetic Resonance to Assess Lyophilized Drug Product Reconstitution.

PURPOSE: Dynamic in-situ proton (1H) magnetic resonance imaging (MRI) and 1H T2-relaxometry experiments are described in an attempt to: (i) understand the physical processes, that occur during the reconstitution of lyophilized bovine serum albumin (BSA) and monoclonal antibody (mAb) proteins; and (ii) objectify the reconstitution time. METHODS: Rapid two-dimensional 1H MRI and diffusion weighted MRI were used to study the temporal changes in solids dissolution and characterise water mass transport characteristics. One-shot T2 relaxation time measurements were also acquired in an attempt to quantify the reconstitution time. Both MRI data and T2-relaxation data were compared to standard visual observations currently adopted by industry. The 1H images were further referenced to MRI calibration data to give quantitative values of protein concentration and, percentage of remaining undissolved solids. RESULTS: An algorithmic analysis of the 1H T2-relaxation data shows it is possible to classify the reconstitution event into three regimes (undissolved, transitional and dissolved). Moreover, a combined analysis of the 2D 1H MRI and 1H T2-relaxation data gives a unique time point that characterises the onset of a reconstituted protein solution within well-defined error bars. These values compared favourably with those from visual observations. Diffusion weighted MRI showed that low concentration BSA and mAb samples showed distinct liquid-liquid phase separation attributed to two liquid layers with significant density differences. CONCLUSIONS: T2 relaxation time distributions (whose interpretation is validated from the 2D 1H MR images) provides a quick and effective framework to build objective, quantitative descriptors of the reconstitution process that facilitate the interpretation of subjective visual observations currently adopted as the standard practice industry.

Practical Considerations for Determination of Glass Transition Temperature of a Maximally Freeze Concentrated Solution.

Glass transition temperature is a unique thermal characteristic of amorphous systems and is associated with changes in physical properties such as heat capacity, viscosity, electrical resistance, and molecular mobility. Glass transition temperature for amorphous solids is referred as (T g), whereas for maximally freeze concentrated solution, the notation is (T g'). This article is focused on the factors affecting determination of T g' for application to lyophilization process design and frozen storage stability. Also, this review provides a perspective on use of various types of solutes in protein formulation and their effect on T g'. Although various analytical techniques are used for determination of T g' based on the changes in physical properties associated with glass transition, the differential scanning calorimetry (DSC) is the most commonly used technique. In this article, an overview of DSC technique is provided along with brief discussion on the alternate analytical techniques for T g' determination. Additionally, challenges associated with T g' determination, using DSC for protein formulations, are discussed. The purpose of this review is to provide a practical industry perspective on determination of T g' for protein formulations as it relates to design and development of lyophilization process and/or for frozen storage; however, a comprehensive review of glass transition temperature (T g, T g'), in general, is outside the scope of this work.

Understanding the impact of mannitol on physical stability and aerosolization of spray-dried protein powders for inhalation.

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.

Survivorship, patient reported outcome and satisfaction following resurfacing and total hip arthroplasty.

Resurfacing (RA) and total hip arthroplasty (THA) are options in the treatment of debilitating hip pathology. 381 patients that had undergone arthroplasty with a BHR RA, ASR RA, metal-on-metal (MoM) THA or ceramic-on-ceramic (CoC) THA were reviewed for satisfaction, function, health and survivorship at a median follow up of 50 months. Significantly lower survivorship for revision and reoperation was observed in the ASR group. The BHR and CoC demonstrated better outcome scores than the ASR (OHS and SAPS) and the BHR better scores than the MoM (OHS and SF12 PCS). In the short to medium term, survivorship and outcomes for the best performing RA (BHR) and THA (CoC) were comparable. There was a non-significant trend towards poorer outcome scores in the MoM THA group.

Oh nuts, they've got a pelvic kidney - a tricky testicular vein embolisation.

Testicular vein embolisation for varicocele is a common interventional procedure performed in predominantly young, healthy males. Cross-sectional imaging is rarely performed for treatment planning and is often not available. In this case report, we describe a case of testicular vein embolisation in an ipsilateral pelvic kidney where cross-sectional imaging aided treatment planning resulting in successful embolisation.

Interventional therapies in acute pulmonary embolus-current trends and future directions.

Venous thromboembolic disease presenting with acute pulmonary embolus (PE) can be treated in a variety of ways from anticoagulation as an outpatient to surgical embolectomy with many new interventional therapies being developed. Mortality in these patients can be as high as 50% and many of these treatments are also considered to be high risk. Early involvement of a multidisciplinary team and patient risk stratification can aid management decisions in these complex patients who can suddenly deteriorate.In this review, we summarise the evidence behind new and developing interventional therapies in the treatment of high and intermediate-high risk PE including catheter-directed thrombolysis, pharmacomechanical thrombolysis, thromboaspiration and the growing role of extracorporeal membrane oxygenation in the stabilisation and management of this cohort of patients.

Effect of Various Silicone Oil And Tungsten Levels on the Stability of a Monoclonal Antibody in Nine Commercially Available Prefilled Syringes.

Prefilled syringes are widely used as a primary container for therapeutic proteins because they are more convenient than glass vials. The stability of biologic molecules can be affected by different syringe materials and techniques, such as silicone oil levels and coating method, amount of tungsten remaining in the glass barrel after using a tungsten pin to create the needle hole, and end of the syringe, which can be Luer locked or pre-staked with a needle. We investigated the impact of these parameters by using a monoclonal antibody to collect the antibody's stability profile and the prefilled syringes' functionality data. Silicone oil levels had no impact on aggregation levels, and particle counts were lowest for silicone oil-free syringes. Functionality performance was similar and did not change throughout all stability time points for all syringe configurations. The break-loose force for Ompi syringes was initially lower and increased over time to align with those of the other configurations, all of which remained well below 25 N. Tungsten contaminants and agitation stress from shipping studies did not impact quality attributes. This work can help guide the development of similar products in prefilled syringes to ensure selection of the primary container that provides adequate stability for the protein, as well as maintain the desired functionality features over the shelf life of the drug product.

Protein Aggregates in Inhaled Biologics: Challenges and Considerations.

Pulmonary delivery is the main route of administration for treatment of local lung diseases. Recently, the interest in delivery of proteins through the pulmonary route for treatment of lung diseases has significantly increased, especially after Covid-19 pandemic. The development of an inhalable protein combines the challenges of inhaled as well as biologic products since protein stability may be compromised during manufacture or delivery. For instance, spray drying is the most common technology for manufacture of inhalable biological particles, however, it imposes shear and thermal stresses which may cause protein unfolding and aggregation post drying. Therefore, protein aggregation should be evaluated for inhaled biologics as it could impact the safety and/or efficacy of the product. While there is extensive knowledge and regulatory guidance on acceptable limits of particles, which inherently include insoluble protein aggregates, in injectable proteins, there is no comparable knowledge for inhaled ones. Moreover, the poor correlation between in vitro setup for analytical testing and the in vivo lung environment limits the predictability of protein aggregation post inhalation. Thus, the purpose of this article is to highlight the major challenges facing the development of inhaled proteins compared to parenteral ones, and to share future thoughts to resolve them.

Impact of controlled ice nucleation and lyoprotectants on nanoparticle stability during Freeze-drying and upon storage.

The freezing step of the lyophilization process can impact nanoparticle stability due to increased particle concentration in the freeze-concentrate. Controlled ice nucleation is a technique to achieve uniform ice crystal formation between vials in the same batch and has attracted increasing attention in pharmaceutical industry. We investigated the impact of controlled ice nucleation on three types of nanoparticles: solid lipid nanoparticles (SLNs), polymeric nanoparticles (PNs), and liposomes. Freezing conditions with different ice nucleation temperatures or freezing rates were employed for freeze-drying all formulations. Both in-process stability and storage stability up to 6 months of all formulations were assessed. Compared with spontaneous ice nucleation, controlled ice nucleation did not cause significant differences in residual moisture and particle size of freeze-dried nanoparticles. The residence time in the freeze-concentrate was a more critical factor influencing the stability of nanoparticles than the ice nucleation temperature. Liposomes freeze-dried with sucrose showed particle size increase during storage regardless of freezing conditions. By replacing sucrose with trehalose, or adding trehalose as a second lyoprotectant, both the physical and chemical stability of freeze-dried liposomes improved. Trehalose was a preferable lyoprotectant than sucrose to better maintain the long-term stability of freeze-dried nanoparticles at room temperature or 40 °C.

Role of arginine salts in preventing freezing-induced increase in subvisible particles in protein formulations.

While arginine hydrochloride (ArgHCl) has emerged as a potential stabilizer of protein drugs in liquid formulations, the purpose of this manuscript was to evaluate its stabilization potential in frozen solutions. The phase behavior of frozen ArgHCl solutions was investigated by differential scanning calorimetry and low temperature powder X-ray diffractometry. The aggregation of ß-galactosidase was evaluated following freeze-thaw cycling in ArgHCl solutions with and without mannitol. ArgHCl (5% w/v) was retained amorphous in frozen aqueous solutions and effectively inhibited protein aggregation even after 5 freeze-thaw cycles. Annealing frozen arginine solution (5% w/v) containing mannitol (10% w/v) induced mannitol crystallization which in turn facilitated crystallization of ArgHCl. The stabilizing effect of ArgHCl was completely lost in the presence of mannitol. Use of alternate arginine salts (aspartate, glutamate, and acetate) allowed selective crystallization of mannitol while arginine was retained amorphous and stabilized the protein.

Qualitative High-Throughput Analysis of Subvisible Particles in Biological Formulations Using Backgrounded Membrane Imaging.

High-throughput analysis of low-volume samples for detection of subvisible particles (SVPs) in biologic formulations remains an unmet need in the pharmaceutical industry. Some commonly used methods, such as light obscuration and microflow imaging, for SVP analysis are not high throughput and require significant amounts of sample volume, which may impede the collection of SVP data when therapeutic protein amounts are limited, typically during early stages of formulation development. We evaluated backgrounded membrane imaging (BMI) as an orthogonal method for SVP analysis and identified critical experimental parameters. Protein concentration, sample viscosity, and membrane coverage area had to be adjusted for each sample, especially those with high protein concentrations. A comparative analysis of particle counts obtained from BMI, light obscuration, and microflow imaging for five protein samples revealed that particle counts obtained with BMI were significantly higher than those acquired with the other two techniques for all particle size categories. BMI could not accurately count particles in protein-containing samples, as the image analysis software could not accurately trace the boundaries of translucent particles. Based on our results, BMI could be used as an orthogonal method for particle characterization when sample material is limited, such as during the early stages of formulation development or screening.

Emerging freeze-drying process development and scale-up issues.

Although several guidelines do exist for freeze-drying process development and scale-up, there are still a number of issues that require additional attention. The objective of this review article is to discuss some emerging process development and scale-up issue with emphasis on effect of load condition and freeze-drying in novel container systems such as syringes, Lyoguard trays, ampoules, and 96-well plates. Understanding the heat and mass transfer under different load conditions and for freeze-drying in these novel container systems will help in developing a robust freeze-drying process which is also easier to scale-up. Further research and development needs in these emerging areas have also been addressed.

Maternal Health in the Transgender Population.

Social acceptance and legal protections for transgender and gender nonconforming patients have increased over the past decade, but significant health care disparities still remain. Such an area of disparity is discussion with and interventions for fertility, contraception, pregnancy, and lactation in TGNC patients. Providing optimal care starts with creating a welcoming and safe environment. Appropriate preconception education includes the effects of gender-affirming therapies (both surgical and nonsurgical) on fertility as well the fertility preservation techniques that are available. However, as gamete retrieval requires natal hormone stimulation, gender dysphoria may be worsened. Thus, these patients should be carefully monitored not only medically, but also with regard to their mental health. In addition to assisted reproductive technologies, protocols exist to aid with induction of lactation as well as discontinuation if desired. As this is a growing field of medicine with limited data available on safety and long-term outcomes, recommendations are for a multidisciplinary team approach to ensure patients' safety and well-being.

Key factors governing the reconstitution time of high concentration lyophilized protein formulations.

Lyophilized protein formulations containing highly concentrated proteins often have long and variable reconstitution times. Reconstitution time is dependent on a number of factors in a complex manner. Furthermore, factors influencing the reconstitution of partially crystalline cakes are reportedly different from those of amorphous cakes. The objectives of this work were to identify the key factors governing reconstitution and understand the mechanisms involved in reconstitution of both amorphous and partially crystalline cakes. Partial crystallinity in the final cake, larger pores and low "concentrated formulation viscosity" (i.e., viscosity near the surface of the dissolving cake) were identified as desirable characteristics for expediting reconstitution. Crystallinity and larger pores dramatically improved wettability and liquid penetration into partially crystalline cakes, ultimately resulting in well dispersed small pieces of partially dissolved cake. The smaller disintegrated cake pieces dissolved faster because of the increased surface area. The amorphous cakes exhibited poorer wettability than partially crystalline cakes. Moreover, the ability of the reconstitution fluid to penetrate the pores, and the resulting cake disintegration was much lower than that observed for partially crystalline cakes. In fact, for some of the amorphous cakes, the reconstitution fluid did not penetrate the cake at all. As a result, the undissolved intact cake or a large cake chunk floated on the reconstitution fluid amidst foam or bubbles generated during reconstitution. Dissolution of the floating cake appeared to proceed via gradual surface erosion where reconstitution time was found to be highly correlated with the viscosity near the surface of the dissolving cake solids. A higher viscosity prolonged reconstitution. Thus, both formulation and processing conditions can be tailored to achieve faster reconstitution. Including a crystallizable excipient proved to be beneficial. Incorporating an annealing step to facilitate crystallization of the crystallizable excipient and to promote larger pores was also found to be advantageous. A viscosity lowering excipient in the formulation could potentially be helpful but needs to be explored further.

Impact of formulation on the quality and stability of freeze-dried nanoparticles.

Freeze-drying is an effective approach to improve the long-term stability of nanomedicines. Lyoprotectants are generally considered as requisite excipients to ensure that the quality of nanoparticles is maintained throughout the freeze-drying process. However, depending on the type of nanoparticles, the needs for lyoprotectants or the challenges they face during freeze-drying may be different. In this study, we compared and identified the impact of freeze-drying on key characteristics of three types of nanoparticles: solid lipid nanoparticles (SLNs), polymeric nanoparticles (PNs), and liposomes. Sucrose, trehalose, and mannitol were added to nanoparticle suspensions before freeze-drying. The same conservative freeze-drying conditions with controlled ice nucleation at -8 °C were employed for all formulations. The collapse temperatures of nanoparticle formulations were found to be the same as those of the lyoprotectant added, except PN formulation. Likely the poly(vinyl alcohol) (PVA) in the formulation induced a higher collapse temperature and retardation of drying of PNs. Freeze-drying of both SLNs and liposomes without lyoprotectants increased particle size and polydispersity, which was resolved by adding amorphous disaccharides. Regardless of the addition of lyoprotectants, freeze-drying did not alter the size of PNs possibly due to the protection from PVA. However, lyoprotectants were still necessary to shorten the reconstitution time and reduce the residual moisture. In conclusion, different types of nanoparticles face distinct challenges for freeze-drying, and lyoprotectants differentially affect various stability and quality attributes of freeze-dried nanoparticles.

Determination of end point of primary drying in freeze-drying process control.

Freeze-drying is a relatively expensive process requiring long processing time, and hence one of the key objectives during freeze-drying process development is to minimize the primary drying time, which is the longest of the three steps in freeze-drying. However, increasing the shelf temperature into secondary drying before all of the ice is removed from the product will likely cause collapse or eutectic melt. Thus, from product quality as well as process economics standpoint, it is very critical to detect the end of primary drying. Experiments were conducted with 5% mannitol and 5% sucrose as model systems. The apparent end point of primary drying was determined by comparative pressure measurement (i.e., Pirani vs. MKS Baratron), dew point, Lyotrack (gas plasma spectroscopy), water concentration from tunable diode laser absorption spectroscopy, condenser pressure, pressure rise test (manometric temperature measurement or variations of this method), and product thermocouples. Vials were pulled out from the drying chamber using a sample thief during late primary and early secondary drying to determine percent residual moisture either gravimetrically or by Karl Fischer, and the cake structure was determined visually for melt-back, collapse, and retention of cake structure at the apparent end point of primary drying (i.e., onset, midpoint, and offset). By far, the Pirani is the best choice of the methods tested for evaluation of the end point of primary drying. Also, it is a batch technique, which is cheap, steam sterilizable, and easy to install without requiring any modification to the existing dryer.

Reconstitution Time for Highly Concentrated Lyophilized Proteins: Role of Formulation and Protein.

Lyophilized protein formulations containing highly concentrated proteins often have long reconstitution times. The goal was to understand the role of formulation in mediating the reconstitution time. Formulation variables such as % total solids, protein concentration, protein-to-sugar ratio, different proteins and inclusion of a crystallizable excipient were investigated for their effect on cake properties influencing reconstitution namely, cake wettability, penetration of reconstitution fluid into the cake, cake disintegration and cake porous structure. Additionally, several measures of viscosity were also evaluated for their effect on reconstitution time. Reconstitution time was primarily influenced by the "concentrated formulation viscosity" with negligible contributions from % total solids and protein concentration. "Concentrated formulation viscosity" was sensitive to both protein-to-sugar ratio and the protein itself. Partial crystallinity in the final cake also expedited reconstitution. Wettability, liquid penetration into the cake, cake disintegration tendency and cake porous structure were found to be invariant for amorphous cakes and did not correlate with reconstitution time. However, these properties were sensitive to the presence of crystallinity and resulted in faster reconstitution at least of the partially crystalline cakes. "Concentrated formulation viscosity" strongly correlated with reconstitution times of amorphous cakes, providing insights on the steps involved in the reconstitution of amorphous formulations.