Role of Poloxamer 188 in Preventing Ice-Surface-Induced Protein Destabilization during Freeze-Thawing.

The phase behavior of poloxamer 188 (P188) in aqueous solutions, characterized by differential scanning calorimetry (DSC) and synchrotron X-ray diffractometry, revealed solute crystallization during both freezing and thawing. Sucrose and trehalose inhibited P188 crystallization during freeze-thawing (FT). While trehalose inhibited P188 crystallization only during cooling, sucrose completely suppressed P188 crystallization during both cooling and heating. Lactate dehydrogenase (LDH) served as a model protein to evaluate the stabilizing effect of P188. The ability of P188, over a concentration range of 0.003-0.800% w/v, to prevent LDH (10 µg/mL) destabilization was evaluated. After five FT cycles, the aggregation behavior (by dynamic light scattering) and activity recovery were evaluated. While LDH alone was sensitive to interfacial stress, P188 at concentrations of ≥0.100% w/v stabilized the protein. However, as the surfactant concentration decreased, protein aggregation after FT increased. The addition of sugar (1.0% w/v; sucrose or trehalose) improved the stabilizing function of P188 at lower concentrations (≤0.010% w/v), possibly due to the inhibition of surfactant crystallization. Based on a comparison with the stabilization effect of polysorbate (both 20 and 80), it was evident that P188 could be a promising alternative surfactant in frozen protein formulations. However, when the surfactant concentration is low, the potential for P188 crystallization and the consequent compromise in its functionality warrant careful consideration.

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.

Frozen storage of proteins: Use of mannitol to generate a homogenous freeze-concentrate.

Therapeutic proteins may be subjected to several freeze-thaw cycles throughout manufacturing and storage. The protein solution composition and the freezing conditions may lead to incomplete ice crystallization in the frozen state. This can also result in freeze-concentrate heterogeneity characterized by multiple glass transition temperatures and protein destabilization. The overall objective was to investigate the potential advantages of including a crystallizing excipient (mannitol) along with a sugar (sucrose or trehalose) for frozen storage. This study showed that the addition of mannitol, a readily crystallizing excipient, facilitated ice crystallization. Inclusion of an isothermal hold during cooling (annealing) maximized the mannitol crystallization and resulted in a homogenous freeze-concentrate of a constant composition characterized by a single glass transition temperature. The role of freezing rate and annealing on both mannitol and ice crystallization were discerned using high intensity synchrotron radiation. The addition of sucrose or trehalose, at an appropriate concentration, stabilized the protein. The mannitol to sugar ratio (3:1 or 1:1, 5 % w/v) was optimized to selectively cause maximal crystallization of mannitol while retaining the sugar amorphous. Human serum albumin (1 mg/mL) in these optimized and annealed compositions did not show any meaningful aggregation, even after multiple freeze-thaw cycles. Thus, in addition to a sugar as a stabilizer, the use of a crystallizing excipient coupled with an annealing step can provide an avenue for frozen storage of proteins.

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.

Phase behavior of poloxamer 188 in frozen aqueous solutions - Influence of processing conditions and cosolutes.

The aim of the study is to investigate the thermal behavior of poloxamer 188 (P188) in binary (P188-water) and ternary (P188-trehalose-water) solutions during freezing and thawing. The thermal behavior of P188 in frozen (binary and ternary) systems was characterized by differential scanning calorimetry (DSC) and low-temperature X-ray powder diffractometry (XPRD) as a complementary technique. The influence of processing conditions (cooling rate, annealing) and a noncrystallizing co-solute (addition of trehalose) on the behavior of P188 was evaluated during freezing as well as thawing. In rapidly cooled (10 °C/min) aqueous binary solutions, P188 (10% w/v) was retained in the amorphous state. At slower cooling rates (0.5-5 °C/min), the extent of crystallization depended on the cooling rate. In ternary P188-trehalose-water systems (P188 4% w/v, trehalose 0-10% w/v), a concentration dependent inhibition of P188 crystallization was observed with increasing trehalose concentration. However, irrespective of trehalose concentration, annealing resulted in P188 crystallization. The presence of trehalose as well as the processing conditions (cooling rate and annealing) influenced the physical state of P188 at different stages of freezing and thawing. As the cooling rate decreased, the extent of P188 crystallization progressively increased. In presence of trehalose (≥4.0% w/v) crystallization of P188 (4.0% w/v) was inhibited and this effect could be reversed by annealing. Depending on the intended application, the physical form of P188 could be modulated, by annealing even in presence of a noncrystallizing solute.

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.

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.

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.

A refined phase diagram of the tert-butanol-water system and implications on lyophilization process optimization of pharmaceuticals.

While water is the solvent of choice for the lyophilization of pharmaceuticals, tert-butyl alcohol (TBA) along with water can confer several advantages including increased solubility of hydrophobic drugs, decreased drying time, improved product stability and reconstitution characteristics. The goal of this work was to generate the phase diagram and determine the eutectic temperature and composition in the "water rich" region (0.0 to 25.0% w/w TBA) of TBA-water mixtures. Solutions of different compositions were frozen and characterized by low temperature differential scanning calorimetry and powder X-ray diffractometry (XRD). The thermal events observed during warming, and their characterization by XRD, enabled the generation of phase boundaries as well as the eutectic temperature and composition. While TBA crystallized as a dihydrate in frozen solutions, on heating, the dihydrate transformed to a heptahydrate. TBA heptahydrate and ice (22.5% w/w TBA) formed a eutectic at ∼-8 °C.