Effect of surfactants on lactate dehydrogenase aqueous solutions: A comparative study of poloxamer 188, polysorbate 20 and 80.

The effect of three commonly used surfactants, poloxamer 188 (P188), polysorbate 20 and 80 (PS20 and PS80), on the stability of a model protein, lactate dehydrogenase (LDH), was compared in aqueous solutions. In the absence of a surfactant, protein solution revealed a gradual decrease in surface tension as a function of time. The addition of surfactant resulted in a rapid decrease in the surface tension. This suggested that the surface behavior was dictated by the surfactant. PS20 and PS80 were more effective than P188 in preventing LDH adsorption on the solution surface. The advantage of polysorbates over P188 was also evident from the higher LDH tetramer recovery after shaking (room temperature, 30 h), especially when the surfactants were used at concentrations ≤ 0.01% w/v. However, PS20 and PS80 accelerated protein unfolding during quiescent storage at 40 °C. Based on circular dichroism results, polysorbates perturbed the tertiary structure of LDH but not the secondary structure, while P188 did not impact the protein structure and stability. Polysorbates were more effective in stabilizing LDH against mechanical stress (shaking), but their adverse effects on protein conformational stability need to be carefully evaluated.

Probing Chemical Equilibrium in Frozen Sodium Phosphate Buffer Solution by 31P Solid-State NMR.

Phosphate buffers are crucial for cryopreservative stability in pharmaceuticals, food processing, biomedical sciences, and biology. However, their freeze concentrates lack quantitative characterization, especially regarding the physicochemical properties of phosphate salt species in equilibrium at subzero temperatures. This study employs 31P solid-state NMR (ssNMR) to analyze frozen sodium phosphate (NaP) solutions, providing insights into phase composition, ionic strength, and pH. For the first time, we have directly quantified phosphate species in frozen NaP buffer, including crystallized disodium phosphate dodecahydrate (Na2HPO4·12H2O) content and the concentrations of H2PO4- and HPO42- in the freeze concentrate. This enabled the calculation of the pH as well as the ionic strength in the freeze concentrate. Trehalose effectively mitigated pH shifts in buffer solutions by preventing the selective crystallization of salt, a spectroscopic phenomenon not previously observed experimentally.

Dual Functionality of Poloxamer 188 in Freeze-Dried Protein Formulations: A Stabilizer in Frozen Solutions and a Bulking Agent in Lyophiles.

Poloxamer 188 (P188) was hypothesized to be a dual functional excipient, (i) a stabilizer in frozen solution to prevent ice-surface-induced protein destabilization and (ii) a bulking agent to provide elegant lyophiles. Based on X-ray diffractometry and differential scanning calorimetry, sucrose, in a concentration-dependent manner, inhibited P188 crystallization during freeze-drying, while trehalose had no such effect. The recovery of lactate dehydrogenase (LDH), the model protein, was evaluated after reconstitution. While low LDH recovery (∼60%) was observed in the lyophiles prepared with P188, the addition of sugar improved the activity recovery to >85%. The secondary structure of LDH in the freeze-dried samples was assessed using infrared spectroscopy, and only moderate structural changes were observed in the lyophiles formulated with P188 and sugar. Thus, P188 can be a promising dual functional excipient in freeze-dried protein formulations. However, P188 alone does not function as a lyoprotectant and needs to be used in combination with a sugar.

Impact of Excipient Extraction and Buffer Exchange on Recombinant Monoclonal Antibody Stability.

The foundation of a biosimilar manufacturer's regulatory filing is the demonstration of analytical and functional similarity between the biosimilar product and the pertinent originator product. The excipients in the formulation may interfere with characterization using typical analytical and functional techniques during this biosimilarity exercise. Consequently, the producers of biosimilar products resort to buffer exchange to isolate the biotherapeutic protein from the drug product formulation. However, the impact that this isolation has on the product stability is not completely known. This study aims to elucidate the extent to which mAb isolation via ultrafiltration-diafiltration-based buffer exchange impacts mAb stability. It has been demonstrated that repeated extraction cycles do result in significant changes in higher-order structure (red-shift of 5.0 nm in fluorescence maxima of buffer exchanged samples) of the mAb and also an increase in formation of basic variants from 19.1 to 26.7% and from 32.3 to 36.9% in extracted innovator and biosimilar Tmab samples, respectively. It was also observed that under certain conditions of tertiary structure disruptions, Tmab could be restabilized depending on formulation composition. Thus, mAb isolation through extraction with buffer exchange impacts the product stability. Based on the observations reported in this paper, we recommend that biosimilar manufacturers take into consideration these effects of excipients on protein stability when performing biosimilarity assessments.

Solid-State NMR Spectroscopy to Probe State and Phase Transitions in Frozen Solutions.

Freezing is commonly encountered during the processing and storage of biomacromolecule products. Therefore, understanding the phase and state transitions in pharmaceutical frozen solutions is crucial for the rational development of biopharmaceuticals. Solid-state nuclear magnetic resonance spectroscopy (ssNMR) was used to analyze solutions containing sodium phosphate buffer, histidine, and trehalose. Upon freezing, crystallization of disodium phosphate hydrogen dodecahydrate (Na2HPO4·12H2O, DPDH) and histidine was identified using 31P and 13C ssNMR, respectively, and confirmed by synchrotron X-ray diffractometry (SXRD). Using histidine as a molecular probe and based on the chemical shifts of atoms of interest, the pH of the freeze concentrate was measured. The unfrozen water content in freeze concentrates was quantified by 1H single pulse experiments. 13C-insensitive nuclei enhancement by polarization transfer (INEPT) and cross-polarization (CP) experiments were used as orthogonal tools to characterize the solutes in a "mobile" and a more "solid-like" state in the freeze-concentrated solutions, respectively. The above analyses were applied to a commercial monoclonal antibody (mAb) formulation of dupilumab. This work further establishes ssNMR spectroscopy as a highly capable biophysical tool to investigate the attributes of biopharmaceuticals and thereby provide insights into process optimization and formulation development.

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.

Understanding the Effect of Nucleation in Amorphous Solid Dispersions through Time-Temperature Transformation.

In an earlier investigation, the critical cooling rate to prevent drug crystallization (CRcrit) during the preparation of nifedipine (NIF) amorphous solid dispersions (ASDs) was determined through a time-temperature transformation (TTT) diagram (Lalge et al. Mol. Pharmaceutics 2023, 20 (3), 1806-1817). The current study aims to use the TTT diagram to determine the critical cooling rate to prevent drug nucleation (CRcrit N) during the preparation of ASDs. ASDs were prepared with each polyvinylpyrrolidone (PVP) and hydroxypropyl methylcellulose acetate succinate (HPMCAS). The dispersions were first stored under conditions promoting nucleation and then heated to the temperature that favors crystallization. The crystallization onset time (tC) was determined by differential scanning calorimetry and synchrotron X-ray diffractometry. TTT diagrams for nucleation were generated, which provided the critical nucleation temperature (50 °C) and the critical cooling rate to avoid nucleation (CRcrit N). The strength of the drug-polymer interactions as well as the polymer concentration affected the CRcrit N, with PVP having a stronger interaction than HPMCAS. The CRcrit of amorphous NIF was ∼17.5 °C/min. The addition of a 20% w/w polymer resulted in CRcrit of ∼0.05 and 0.2 °C/min and CRcrit N of ∼4.1 and 8.1 °C/min for the dispersions prepared with PVP and HPMCAS, respectively.

Drug Phase Transformation and Water Redistribution during Continuous Tablet Manufacturing: A Case Study of Carbamazepine Dihydrate.

In recent years, continuous tablet manufacturing technology has been used to obtain regulatory approval of several new drug products. While a significant fraction of active pharmaceutical ingredients exists as hydrates (wherein water is incorporated stoichiometrically in the crystal lattice), the impact of processing conditions and formulation composition on the dehydration behavior of hydrates during continuous manufacturing has not been investigated. Using powder X-ray diffractometry, we monitored the dehydration kinetics of carbamazepine dihydrate in formulations containing dibasic calcium phosphate, anhydrous (DCPA), mannitol, or microcrystalline cellulose. The combined effect of nitrogen flow and vigorous mixing during the continuous mixing stage of tablet manufacture facilitated API dehydration. Dehydration was rapid and most pronounced in the presence of DCPA. The dehydration product, amorphous anhydrous carbamazepine, sorbed a significant fraction of the water released by dehydration. Thus, the dehydration process resulted in a redistribution of water in the powder blend. The unintended formation of an amorphous dehydrated phase, which tends to be much more reactive than its crystalline counterparts, is of concern and warrants further investigation.

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.

Implications of Drug-Polymer Interactions on Time-Temperature-Transformation: A Tool to Assess the Crystallization Propensity in Amorphous Solid Dispersions.

The critical cooling rate (CRcrit) to prevent drug crystallization during the preparation of nifedipine amorphous solid dispersions (ASDs) was determined through the time-temperature-transformation (TTT) diagram. ASDs were prepared with polyvinylpyrrolidone, hydroxypropylmethyl cellulose acetate succinate, and poly(acrylic acid). ASDs were subjected to isothermal crystallization over a wide temperature range, and the time and temperature dependence of nifedipine crystallization onset time (tC) was determined by differential scanning calorimetry (DSC) and synchrotron X-ray diffractometry. TTT diagrams were generated for ASDs, which provided the CRcrit for the dispersions prepared with each polymer. The observed differences in CRcrit could be explained in terms of differences in the strength of interactions. Stronger drug-polymer interactions led to longer tC and decreased CRcrit. The effect of polymer concentrations (4-20% w/w) was also influenced by the strength of the interaction. The CRcrit of amorphous NIF was ∼17.5 °C/min. Addition of 20% w/w polymer resulted in a CRcrit of ∼0.05, 0.2, and 11 °C/min for the dispersions prepared with PVP, HPMCAS, and PAA, respectively.

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.

Succinate Buffer in Biologics Products: Real-world Formulation Considerations, Processing Risks and Mitigation Strategies.

The succinic acid/succinate system has an excellent buffering capacity at acidic pH values (4.5-6.0), promising to be a buffer of choice for biologics having slightly acidic to basic isoelectric points (pI 6 - 9). However, its prevalence in drug products is limited due to the propensity (risk) of its components to crystallize during freezing and the consequent shift in the pH which might affect the product stability. Most of these previous assessments have been performed under operational conditions that do not simulate typical drug product processing conditions. In this work, we have characterized the physicochemical behavior of succinate formulations under representative pharmaceutical conditions. Our results indicate that the pH increases by ∼ 1.2 units in 25 mM and 250 mM succinate buffers at pharmaceutically relevant freezing conditions. X-ray diffractometry studies revealed selective crystallization of monosodium succinate, which is posed as the causative mechanism. This salt crystallization was not observed in the presence of 2% w/v sucrose, suggesting that this pH shift can be mitigated by including sucrose in the formulation. Additionally, three monoclonal antibodies (mAbs) that represent different IgG subtypes and span a range of pIs (5.9 - 8.8) were formulated with succinate and sucrose and subjected to freeze-thaw, frozen storage and lyophilization. No detrimental impact on quality attributes (QA) such as high molecular weight (HMW) species, turbidity, alteration in protein concentration and sub-visible particles, was observed of any of the mAbs tested. Lastly, drug formulations lyophilized in succinate buffer with sucrose demonstrated acceptable QA profiles upon accelerated kinetic storage stability, supporting the use of succinate buffers in mAb drug products.

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.

Design of Ternary Amorphous Solid Dispersions for Enhanced Dissolution of Drug Combinations.

Using sulfamethoxazole (SMZ) and trimethoprim (TMP) as model drugs, we designed amorphous solid dispersions (ASDs) for the simultaneous solubility enhancement of two active pharmaceutical ingredients (APIs) by exploiting the drug-drug and drug-polymer interactions. In order to make this approach broadly applicable and over a wide dose range, a mixture of SMZ and TMP at weight ratios of 5:1 and 1:5 (w/w) were formulated into ternary ASDs. Depending on the dose ratio of the two drugs, the polymer used was either an aminoalkyl methacrylate copolymer (Eudragit, EDE) or polyacrylic acid. The drug-drug and drug-polymer interactions were characterized to be ionic by infrared and solid-state nuclear magnetic resonance spectroscopy. The interactions resulted in a substantial reduction in molecular mobility, evident from the increase in the structural relaxation time determined by dielectric spectroscopy. The drug-drug interaction resulted in ∼3 orders of magnitude reduction in molecular mobility. The addition of a polymer led to a further decrease in molecular mobility of up to 4 orders of magnitude. The strength of intermolecular interactions was also estimated from the glass transition temperatures of the ASDs obtained by differential scanning calorimetry. The strong intermolecular interactions yielded highly stable ASDs with no evidence of crystallization, both at elevated temperatures and under accelerated storage conditions (40 °C/75% relative humidity; 6 weeks). The dissolution performances of the ASDs were evaluated using the area under the curve (AUC) obtained from the concentration-time profiles under the non-sink condition. SMZ and TMP in their ternary ASDs, when compared with their crystalline counterparts, exhibited up to 6.4- and 4.6-fold increases in AUC, respectively. Importantly, the synchronized release of the two drugs was observed, a desirable attribute in synergistic formulations. A single-phase ternary ASD, stabilized by drug-drug and drug-polymer interactions, is likely responsible for the unique release profile.

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.

Dual Functionality of Bile Acid: Physical Stabilization of Drugs in the Amorphous Form and Solubility Enhancement in Solution.

Drugs containing an amino aromatic nitrogen moiety were stabilized in the amorphous form by the surfactant cholic acid (CA). Coamorphous systems of lamotrigine (LAM), pyrimethamine (PYR), and trimethoprim (TRI) were each prepared with CA. Drug-CA interactions, investigated by IR and solid-sate NMR spectroscopy, revealed deprotonation of the carboxylic acid group in CA and the protonation of the most basic nitrogen of the drug. The coamorphous systems exhibited exceptional physical stability and resisted crystallization at (i) elevated temperatures (>100 °C) and (ii) accelerated storage conditions, 40 °C/75% relative humidity for 15 months. The dissolution performance of each coamorphous system was compared with the respective crystalline drug based on the area under the curve (AUC) of the concentration-time profiles. A 25-fold increase in AUC was observed in the PYR-CA coamorphous system. The solubility enhancement is attributed not only due to drug amorphization but also due to solubilization by CA. The supramolecular synthon approach, through a drug-CA interaction, yielded physically stable coamorphous systems with enhanced aqueous drug solubility.

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.

Crystallization Propensity of Amorphous Pharmaceuticals: Kinetics and Thermodynamics.

Four model compounds, nifedipine, indomethacin, felodipine, and ketoconazole, all with nearly identical glass transition temperatures, were chosen to study the effects of thermodynamics and molecular mobility on their crystallization propensities. The time and temperature dependence of the crystallization induction time of each compound was determined by differential scanning calorimetry (DSC) and enabled the generation of their time-temperature-transformation (TTT) diagrams. The relaxation times (τα) were measured by dielectric spectroscopy, and the Gibbs free energy (ΔG) and entropy (ΔS) difference between the crystalline and amorphous states were obtained by DSC. The temperature dependence of the crystallization induction time (τ0(T)) is a function of the thermodynamic activation barrier and the frequency of "attempted jumps" (1/τα(T)) to overcome the barrier. Even though the four model compounds exhibited very similar molecular mobility (relaxation time) over a wide range of temperatures, their crystallization propensities were very different. The observed difference in crystallization propensity was explained in terms of the difference in the thermodynamic barrier, and it is correlated to the empirical relation (TΔS3)/ΔG2.

Use of Atomic Force Microscopy (AFM) to monitor surface crystallization in caffeine-oxalic acid (CAFOXA) cocrystal compacts.

Our objective was to monitor the surface crystallization in disordered caffeine-oxalic acid (CAFOXA) cocrystals following exposure to elevated water vapor pressure. This was accomplished using atomic force microscopy (AFM). Disorder was induced in the cocrystal particles by the common pharmaceutical unit operations of milling and compaction. The 'activated' solid, upon exposure to elevated water vapor pressure, had a high propensity to sorb water. This led to a rise in molecular mobility and the surface underwent rapid crystallization to form needle shaped crystals of CAFOXA. Using AFM height and phase imaging, we were able to directly visualize phase transformations on the compact surface. The milled compacts exhibited higher processing induced disorder than the unmilled compacts, thereby accelerating the surface recrystallization.

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.