Enabling Efficient Design of Biological Formulations Through Advanced Characterization.

The present review summarizes the use of differential scanning calorimetry (DSC) and scattering techniques in the context of protein formulation design and characterization. The scattering techniques include wide angle X-ray diffractometry (XRD), small-angle neutron scattering (SANS) and small-angle X-ray scattering (SAXS). While DSC is valuable for understanding thermal behavior of the excipients, XRD provides critical information about physical state of solutes during freezing, annealing and in the final lyophile. However, as these techniques lack the sensitivity to detect biomolecule-related transitions, complementary characterization techniques such as small-angle scattering can provide valuable insights.

Professor Raj Suryanarayanan: Scientist, Educator, Mentor, Family Man and Giant in Pharmaceutical Research.

This special edition of the Journal of Pharmaceutical Sciences is dedicated to Professor Raj Suryanarayanan (Professor and William & Mildred Peters Endowed Chair, University of Minnesota, School of Pharmacy) and honors his extensive and distinguished career as a scientist, educator and mentor. The goal of this commentary is to provide an overview of Professor Suryanarayanan's noteworthy career path and summarize his key research contributions. The commentary concludes with the personal summaries by guest editors.

Mannitol as an Excipient for Lyophilized Injectable Formulations.

The review summarizes the current state of knowledge of mannitol as an excipient in lyophilized injectable small and large molecule formulations. When compared with glycine, the physicochemical properties of mannitol make it a desirable and preferred bulking agent. Though mannitol is a popular bulking agent in freeze-dried formulations, its use may pose certain challenges such as vial breakage or its existence as a metastable crystalline hemihydrate in the final cake, necessitating appropriate mitigation strategies. The understanding of the phase behavior of mannitol in aqueous systems, during the various stages of freeze-drying, can be critical for the optimization of freeze-drying cycle parameters in multi-component formulations. Finally, using a decision tree as a guiding tool, we demonstrate the use of orthogonal techniques for attaining a stable and cost-effective lyophilized drug product containing mannitol.

Mannitol hemihydrate in lyophilized protein formulations:Impact of its dehydration during storage on sucrose crystallinity and protein stability.

The high propensity of mannitol to crystallize in frozen solutions along with its high eutectic temperature enabling higher primary drying temperatures makes it a good bulking agent. In protein formulations, addition of a sugar (sucrose) that has the ability to remain amorphous throughout processing as well as storage is imperative to retain the protein in its native state. It is well known that in the presence of amorphous excipients and protein, mannitol can crystallize as a mixture of anhydrous polymorphs - α-, ß- and δ-forms and a hemihydrate form [mannitol hemihydrate (MHH); C6H14O6·0.5H2O]. The conditions of formation of MHH due to processing and formulation variables are well established in the literature. However, MHH's dehydration kinetics on storage and its impact on the stability of a protein has not been systematically evaluated. The overall objective was to identify conditions (temperature and humidity) at which MHH can dehydrate on storage and the consequences of the release of associated water on sucrose phase behavior and protein stability. In a mannitol-sucrose-protein lyophile, the purpose of this study was (i) to investigate the dehydration behavior of MHH (ii) to determine the influence of dehydration on sucrose crystallization and (iii) the effect of moisture released due to MHH dehydration on model protein (Bovine serum albumin, BSA or Human serum albumin, HSA) aggregation. MHH dehydration and sucrose crystallization was observed in cases where the relative humidity was ≥ 55% (open vials). A relative humidity of ≤ 33% RH prevented MHH dehydration while retaining sucrose amorphous. No protein aggregation was observed irrespective of presence of MHH or its dehydration.

Reversible Self-Association in Lactate Dehydrogenase during Freeze-Thaw in Buffered Solutions Using Neutron Scattering.

The aims of this work were to evaluate the effect of freezing and thawing stresses on lactate dehydrogenase (LDH) stability under three conditions. (i) In a solution buffered with sodium phosphate (NaP; 10 and 100 mM). The selective crystallization of disodium hydrogen phosphate during freezing caused a pronounced pH shift. (ii) In a solution buffered with histidine, where there was no pH shift due to buffer salt crystallization. (iii) At different concentrations of LDH so as to determine the self-stabilizing ability of LDH. The change in LDH tetrameric conformation was measured by small-angle neutron scattering (SANS). The pH of the phosphate buffer solutions was monitored as a function of temperature to quantify the pH shift. The conditions of buffer component crystallization from solution were identified using low-temperature X-ray diffractometry. Dynamic light scattering (DLS) enabled us to determine the effect of freeze-thawing on the protein aggregation behavior. LDH, at a high concentration (1000 µg/mL; buffer concentration 10 mM), has a pronounced self-stabilizing effect and did not aggregate after five freeze-thaw cycles. At lower LDH concentrations (10 and 100 µg/mL), only with the selection of an appropriate buffer, irreversible aggregation could be avoided. While SANS provided qualitative information with respect to protein conformation, the insights from DLS were quantitative with respect to the particle size of the aggregates. SANS is the only technique which can characterize the protein both in the frozen and thawed states.

Stabilizers and their interaction with formulation components in frozen and freeze-dried protein formulations.

This review aims to provide an overview of the current knowledge on protein stabilization during freezing and freeze-drying in relation to stress conditions commonly encountered during these processes. The traditional as well as refined mechanisms by which excipients may stabilize proteins are presented. These stabilizers encompass a wide variety of compounds including sugars, sugar alcohols, amino acids, surfactants, buffers and polymers. The rational selection of excipients for use in frozen and freeze-dried protein formulations is presented. Lyophilized protein formulations are generally multicomponent systems, providing numerous possibilities of excipient-excipient and protein-excipient interactions. The interplay of different formulation components on the protein stability and excipient functionality in the frozen and freeze-dried systems are reviewed, with discussion of representative examples of such interactions.

t-Butanol Enables Dual Functionality of Mannitol: A Cryoprotectant in Frozen Systems and Bulking Agent in Freeze-Dried Formulations.

The effect of tertiary butyl alcohol (TBA) as a cosolvent on the phase behavior of mannitol in frozen and freeze-dried systems was characterized using differential scanning calorimetry (DSC) and X-ray diffractometry (XRD; laboratory and synchrotron sources). Solutions of mannitol (2 and 5% w/w) in TBA-water systems of different compositions (5 to 30% w/w TBA) were characterized, both during cooling and warming using DSC and XRD. At and below the TBA-water eutectic composition (22.5% w/w TBA), mannitol crystallization was completely inhibited in the frozen state, while it crystallized as anhydrous δ-mannitol in the final lyophile. The presence of mannitol did not affect the phase behavior of TBA. The ability of mannitol to serve as a cryoprotectant in frozen solutions, and as a bulking agent in final lyophile was evaluated using human serum albumin (HSA) as a model protein. When HSA in a TBA (5% w/w)-water solution containing mannitol (2% w/w) was freeze-thawed or freeze-dried, there was no evidence of HSA aggregation. Thus, when TBA was used as a cosolvent, mannitol exhibited dual functionality, serving as a cryoprotectant in frozen solutions and as a bulking agent in the final lyophile.

Anomalous behavior of mannitol hemihydrate: Implications on sucrose crystallization in colyophilized systems.

When solutions containing mannitol and sucrose are freeze-dried, depending on the processing conditions and the formulation composition, mannitol can crystallize in the anhydrous form, as mannitol hemihydrate (MHH; C6H14O6·0.5H2O) or as a mixture of the two. The retention of MHH in the final lyophile, and its dehydration during product storage could lead to instability of the final drug product. Our aim was to determine the influence of water vapor pressure on the kinetics of MHH dehydration and the implications on the physical stability of sucrose. Therefore, the lyophiles were exposed to a range of relative humidities (RH) and the kinetics of MHH dehydration and sucrose crystallization were monitored by X-ray diffractometry. A second set of vials (rubber stoppers fitted with humidity/temperature sensor) were stored at 40 °C, the headspace RH was continually recorded and water content was determined by Karl Fischer titrimetry. The dehydration rate of MHH increased as a function of water vapor pressure, an anomalous behavior explained by the Smith-Topley effect. An increase in headspace RH and decrease in lyophile water content in sealed vials attributed to MHH dehydration, eventually triggered sucrose crystallization. There was also evidence of moisture transfer from the lyophile to the rubber stoppers.

Asymmetric in-plane shear behavior of isolated cadaveric lumbar facet capsular ligaments: Implications for subject specific biomechanical models.

The facet capsular ligaments (FCLs) flank the spinous process on the posterior aspect of the spine. The lumbar FCL is collagenous, with collagen fibers aligned primarily bone-to-bone (medial-lateral) and experiences significant shear, especially during spinal flexion and extension. We characterized the mechanical response of the lumbar FCL to in-plane shear, and we evaluated that response in the context of the fiber architecture. In-plane shear tests with both positive and negative shear (i.e., corresponding to flexion and to extension) were performed on eight cadaveric human L4-L5 FCLs. Our most striking observation was subject-dependent asymmetry in the response. All samples showed a toe region of low stiffness, transitioning to greater stiffness at higher strains, for both shear directions. Different samples showed profoundly different transition strains, with some samples stiffening more rapidly in positive shear and some in negative shear. This unpredictable asymmetry, which did not correlate with age, side, or degeneration state, suggesting that collagen fibers in the FCL are sometimes aligned at a slight positive angle from the bone-to-bone axis and sometimes at a negative angle. Fitting the experimental data to a fiber-composite-based finite element model supported this idea, yielding optimal fits with positive or negative off-axis fiber directions (-40° to +40°). Subsequent examination of selected FCLs by small-angle x-ray scattering (SAXS) showed a similar variability in fiber direction. We conclude that small individual differences in lumbar FCL architecture may have a significant effect on lumbar FCL mechanics, especially at moderate strains.

Role of Lattice Disorder in Water-Mediated Dissociation of Pharmaceutical Cocrystal Systems.

Our objective is to mechanistically understand the implications of processing-induced lattice disorder on  the stability of pharmaceutical cocrystals. Caffeine-oxalic acid (CAFOXA) and dicalcium phosphate anhydrate (DCPA) were the model cocrystal (drug) and excipient, respectively. Cocrystal-excipient mixtures were milled for short times (≤2 min) and stored at room temperature (RT)/75% RH. Milling-induced lattice disorder was quantified using powder X-ray diffractometry and gravimetric water sorption. Milling for even 10 s resulted in measurable disorder and an attendant tendency of the solid to sorb water. This was followed by  cocrystal-excipient interaction leading to dissociation. The proposed mechanism of cocrystal dissociation entails the following sequence: sorption of water by disordered regions, dissolution of CAFOXA and DCPA in the sorbed water, followed by proton transfer from the coformer (oxalic acid) to DCPA, and the formation of hydrates of caffeine and calcium oxalate. As such, CAFOXA is a robust cocrystal, stable even under elevated humidity conditions (RT/98% RH). However, in a drug product environment, routine pharmaceutical processing steps such as milling and compaction have the potential to induce sufficient disorder to render it unstable.

Effect of processing conditions and excipients on dehydration kinetics of sodium naproxen hydrate in formulation.

The manufacture of oral dosage form may induce changes in the physical form of an active pharmaceutical ingredient. One such example includes formation of hydrate during granulation followed by the reverse transition to the anhydrous form during drying. We used naproxen sodium dihydrate (DH) as the model compound and studied its dehydration at elevated temperature under different processing conditions, (i) in ambient air, (ii) in flow of inert gas (iii) under low pressure environment, and (iv) under 'high' pressure in closed environments. In situ variable temperature X-ray diffraction was used to monitor kinetics of phase transformation under these processing conditions. The DH dehydration was fastest under the flow of inert gas and slowest in closed environment. Polyvinyl pyrrolidone (PVP) and microcrystalline cellulose (MCC), commonly used hygroscopic solids, were used as the model excipients to monitor influence of excipients in modulating DH dehydration behavior under different processing conditions. Both the excipients altered the kinetics as well as the extent of DH dehydration, with PVP delaying and MCC facilitating the transformation under all processing conditions studied. The results indicate that the physical form of API, such as hydrate or anhydrous in the present case, in the formulation may be modulated by rational excipient selection.

Compression-Induced Polymorphic Transformation in Tablets: Role of Shear Stress and Development of Mitigation Strategies.

Our goals were to evaluate the effects of (i) hydrostatic pressure alone and (ii) its combined effect with shear stress during compaction, on the polymorphic transformation (form C → A) of a model drug, chlorpropamide. The powder was either subjected to hydrostatic pressure in a pressure vessel or compressed in a tablet press, at pressures ranging from 25 to 150 MPa. The overall extent of phase transformation was determined by powder X-ray diffractometry, whereas 2D-X-ray diffractometry enabled quantification of the spatial distribution of phase composition in tablets. Irrespective of the pressure, the extent of transformation following compaction was higher than that because of hydrostatic pressure alone, the difference attributed to the contribution of shear stress experienced during compaction. At a compression pressure of 25 MPa, there was a pronounced gradient in the extent of phase transformation when monitored from radial tablet surface to core. This gradient decreased with increase in compression pressure. Four approaches were attempted to minimize the effect of compression-induced phase transformation: (a) site-specific lubrication, (b) use of a viscoelastic excipient, (c) ceramic-lined die, and (d) use of cavity tablet. The ceramic-lined die coupled with site-specific lubrication was most effective in minimizing the extent of compression-induced phase transformation.

Applications of Powder X-Ray Diffraction in Small Molecule Pharmaceuticals: Achievements and Aspirations.

Since the discovery of X-ray diffraction and its potential to elucidate crystal symmetry, powder X-ray diffraction has found diverse applications in the field of pharmaceutical sciences. This review summarizes significant achievements of the technique during various stages of dosage form development. Improved understanding of the principle involved and development of automated hardware and reliable software have led to increased instrumental sensitivity and improved data analysis. These advances continue to expand the applications of powder X-ray diffraction to emerging research fields such as amorphous systems, mechanistic understanding of phase transformations, and "Quality by Design" in formulation development.

Evaluation of novel formulations of d-ß-hydroxybutyrate and melatonin in a rat model of hemorrhagic shock.

d-ß-hydroxybutyrate and melatonin (BHB/MLT) infusion improves survival in hemorrhagic shock models. The original BHB/MLT formulation contains dimethyl sulfoxide (DMSO) to increase melatonin solubility. We formulated BHB/MLT solutions wherein DMSO was replaced either with 10% polyvinylpyrrolidone (BHB/MLT/PVP) or with 5% hydroxypropyl-ß-cyclodextrin/2.5% PVP/2.5% polyethylene glycol 400 (BHB/MLT/CD). Safety and efficacy of the new and the original BHB/MLT solution were tested in a lethal rat hemorrhagic shock model, with seven groups: 1) sham, 2) shock, untreated, 3) shock, lactated Ringer's solution (LR), 4) shock, 4 M BHB/MLT/DMSO, 5) shock, 2 M BHB/MLT/DMSO, 6) shock, BHB/MLT/PVP and 7) shock, BHB/MLT/CD. BHB/MLT/DMSO was given at full strength and 1:1 dilution to match the concentration of the novel formulations. Rats were anesthetized, instrumented, and 40% of the total blood volume was withdrawn in three steps, followed by four-hour long shock. Treatment boluses were infused half-way throughout hemorrhage. Survival was highest in BHB/MLT/CD-treated rats (8/10), followed by the BHB/MLT/PVP (6/10), 4 M BHB/MLT/DMSO (5/10) or 2 M BHB/MLT/DMSO (5/10), LR (3/10) and the untreated group (0/11). Survival did not differ significantly between BHB/MLT groups (p > 0.05), but was significantly higher in BHB/MLT/CD than in LR-treated animals (p = 0.018). BHB/MLT/PVP and BHB/MLT/CD constitute promising candidates for clinical hemorrhagic shock treatment.

Effect of Formulation and Process Parameters on the Disproportionation of Indomethacin Sodium in Buffered Lyophilized Formulations.

PURPOSE: (i) To investigate buffer salt crystallization and the consequent pH shifts during the freezing stage of the lyophilization of indomethacin sodium (IMCNa) in aqueous sodium phosphate buffer. (ii) To determine the effect of pH shift on the disproportionation of IMCNa in lyophilized formulations. METHODS: Prelyophilization solutions containing IMCNa in sodium phosphate buffer, at initial buffer concentrations ranging from 10 to 100 mM (pH 7.0), and at IMCNa concentrations of 5, 10 & 15 mg/ml, were investigated. Their phase behavior during cooling was monitored by low temperature X- ray diffractometry (XRD), differential scanning calorimetry (DSC) and pH measurements. The final lyophiles were characterized by infrared spectroscopy (IR) and XRD. RESULTS: Upon cooling to -25°C, pronounced pH shifts were observed only in IMCNa buffered solutions containing high initial buffer concentration (100 mM), due to crystallization of Na2HPO4. 12H2O. In the final lyophiles, disproportionation of IMCNa to the free acid (IMC) was observed in systems with buffer concentrations ≥50 mM, but not low buffer concentration (10 mM). At intermediate buffer concentrations (35 & 20 mM) the disproportionation depended on IMCNa concentration. The initial concentrations of both buffer and IMCNa influenced the buffer crystallization. CONCLUSIONS: During freeze drying, selective crystallization of a buffer component and the consequent pH shift can cause disproportionation of IMCNa. This can prolong the reconstitution time or retain particles of the poorly soluble free acid in the reconstituted solution.

Estimation of Drug Particle Size in Intact Tablets by 2-Dimensional X-Ray Diffractometry.

The average grain size of a crystalline material can be determined from the γ-profile of Debye rings in 2-dimensional X-ray diffraction frames. Our objectives were to: (1) validate the method for organic powders and use it to determine the grain size in intact tablets, and (2) demonstrate the pharmaceutical application of this technique by determining the grain size of the active pharmaceutical ingredient in marketed formulations. Six sieve fractions of sucrose were prepared and the particle size distribution was confirmed by laser diffraction. Their average grain size was determined from the 2-dimensional X-ray diffraction frames by the γ-profile method. For particles <90 µm (based on sieving), the average particle size determined by the 3 methods were in good agreement. When these particles were compressed, there was no discernible change in the sucrose grain size in tablets. When the particles were >250 µm, compression resulted in a mixture of large grains and fine powder. The grain size of acetaminophen in 11 marketed tablet formulations was determined to be either ∼35 µm or ∼80 µm. This nondestructive technique can therefore be potentially useful to estimate the grain size of crystalline formulation components in intact tablets.

Development and in vivo evaluation of a novel lyophilized formulation for the treatment of hemorrhagic shock.

Hemorrhagic shock, caused by trauma, is a leading cause of preventable death. A combination treatment of d-ß-hydroxybutyrate (BHB) and melatonin (MLT), in dimethyl sulfoxide - water, increased survival. A freeze-dried BHB-MLT formulation, with a short reconstitution time, has been developed. This intravenous formulation, prepared with an aqueous vehicle, completely eliminated dimethyl sulfoxide, thereby avoiding the potential problems associated with this solvent. The poor aqueous solubility of MLT necessitated the use of polyvinylpyrrolidine (PVP) as a complexing agent. Thus the prelyophilization solution contained BHB (2 M), MLT (21.5 mM) and PVP (40 mM). Using a combination of low-temperature X-ray diffractometry and thermal analysis, the lyophilization process parameters were optimized. Infra-red spectra revealed hydrogen bonding interaction between PVP and MLT, while BHB crystallized as BHB.0.25 H2O in the final lyophile. The formulation improved survival in a rat model of hemorrhagic shock. Based on the increase in rate of survival and longer survival time compared to untreated animals, we conclude that this formulation can serve as a promising first line of treatment for hemorrhagic shock.

Construction and Validation of Binary Phase Diagram for Amorphous Solid Dispersion Using Flory-Huggins Theory.

Drug-polymer miscibility is one of the fundamental prerequisite for the successful design and development of amorphous solid dispersion formulation. The purpose of the present work is to provide an example of the theoretical estimation of drug-polymer miscibility and solubility on the basis of Flory-Huggins (F-H) theory and experimental validation of the phase diagram. The F-H interaction parameter, χ d-p, of model system, aceclofenac and Soluplus, was estimated by two methods: by melting point depression of drug in presence of different polymer fractions and by Hildebrand and Scott solubility parameter calculations. The simplified relationship between the F-H interaction parameter and temperature was established. This enabled us to generate free energy of mixing (ΔG mix) curves for varying drug-polymer compositions at different temperatures and finally the spinodal curve. The predicted behavior of the binary system was evaluated through X-ray diffraction, differential scanning calorimetry, and in vitro dissolution studies. The results suggest possibility of employing interaction parameter as preliminary tool for the estimation of drug-polymer miscibility.

Recent advances in the characterization of amorphous pharmaceuticals by X-ray diffractometry.

For poorly water soluble drugs, the amorphous state provides an avenue to enhance oral bioavailability. The preparation method, in addition to sample history, can dictate the nature and the stability of the amorphous phase. Conventionally, X-ray powder diffractometry is of limited utility for characterization, but structural insights into amorphous and nanocrystalline materials have been enabled by coupling X-ray total scattering with the pair distribution function. This has shown great promise for fingerprinting, quantification, and even modeling of amorphous pharmaceutical systems. A consequence of the physical instability of amorphous phases is their crystallization propensity, and recent instrumental advances have substantially enhanced our ability to detect and quantify crystallization in a variety of complex matrices. The International Centre for Diffraction Data has a collection of the X-ray diffraction patterns of amorphous drugs and excipients and, based on the available supporting information, provides a quality mark of the data.

Salt formation during freeze-drying--an approach to enhance indomethacin dissolution.

PURPOSE: (i) Prepare a freeze-dried injectable indomethacin (IMC) dosage form. (ii) Convert IMC to its tris salt during freeze-drying so as to facilitate rapid dissolution (reconstitution). (iii) Modulate salt crystallinity by annealing the frozen solution. METHODS: Aqueous IMC solutions buffered with tris were freeze dried, with or without annealing the frozen solutions. The lyophiles were characterized by X-ray diffractometry, differential scanning calorimetry and infra-red spectroscopy and also subjected to water sorption and dissolution studies. RESULTS: Based on IR spectroscopy, the final lyophile was confirmed to contain the IMC tris salt. In the absence of annealing, the lyophile was X-ray amorphous with a glass transition temperature of 19°C. Annealing the frozen solutions caused a substantial increase in lyophile crystallinity. Interestingly, both the amorphous and partially crystalline lyophiles dissolved "instantaneously" and completely in the dissolution medium. In contrast, the crystalline IMC as well as its physical mixture with tris exhibited much slower dissolution with ~ 50% drug dissolved in 30 min. CONCLUSION: In situ IMC tris salt formation resulted in an elegant lyophile with a very short reconstitution time. Tris served two roles - as a buffer in the prelyophilization solution and as the counterion for the salt in the final lyophile. This approach for solubility enhancement could be extended to other acidic drugs wherein salt formation was observed during freeze-drying.