pH Stability Profile Research Articles

Improvement of the catalytic performance of chitosanase Csn-PD from Paenibacillus dendritiformis by semi-rational design

Chitooligosaccharides (COS) possess versatile functional properties that have found extensive applications across various fields. Chitosanase can specifically hydrolyze β-1,4 glycosidic bonds in chitosan to produce COS. In this study, Csn-PD, a glycoside hydrolase family 46 chitosanase from Paenibacillus dendritiformis, which produces (GlcN)2 as its main product, was rationally redesigned aiming to improve its catalytic performance. Based on the results of molecular docking analysis and multiple sequence alignment, four amino acid residues in Csn-PD (I101, T120, T220, and Y259) were pinpointed for targeted mutations. Beneficial mutations in terms of enhanced catalytic activity were then combined by site-directed mutagenesis. Notably, the most promising variant, Csn-PDT6 (Csn-PD I101M/T120E/T220G), exhibited an impressive eight-fold surge in activity compared to the wild-type Csn-PD. This heightened enzymatic activity was complemented by an enhanced pH stability profile. A compelling feature of Csn-PDT6 is its preservation of the hydrolytic product profile observed in Csn-PD. This characteristic further accentuates its candidacy for the targeted production of (GlcN)2. The success of our strategic approach is vividly illustrated by the significant improvements achieved in the catalytic performance of the chitosanase, encompassing both its activity and stability. These developments offer a valuable model that may have implications for the semi-rational design of other enzymes.

Thermodynamics, kinetics and optimization of catalytic behavior of polyacrylamide-entrapped carboxymethyl cellulase (CMCase) for prospective industrial use.

In the current study, kinetic and thermodynamic parameters of free and polyacrylamide-immobilized CMCase were analyzed. The maximum immobilization yield of 34 ± 1.7% was achieved at 11% acrylamide. The enthalpy of activation (ΔH) of free and immobilized enzyme was found to be 13.61 and 0.29kJmol-1, respectively. Irreversible inactivation energy of free and immobilized CMCase was 96.43 and 99.01kJmol-1, respectively. Similarly, the enthalpy of deactivation (ΔHd) values for free and immobilized enzyme were found to be in the range of 93.51-93.76kJmol-1 and 96.08-96.33kJmol-1, respectively. Michaelis-Menten constant (Km) increased from 1.267 ± 0.06 to 1.5891 ± 0.07mgml-1 and the maximum reaction rate (Vmax) value decreased (8319.47 ± 416 to 5643.34 ± 282 U ml-1min-1) after immobilization. Due to wide pH and temperature stability profile with sufficient reusing efficiency up to three successive cycles, the immobilized CMCase might be useful for various industrial processes.

Optimization of Inulin Hydrolysis by Penicillium lanosocoeruleum Inulinases and Efficient Conversion Into Polyhydroxyalkanoates.

Inulin, a polydisperse fructan found as a common storage polysaccharide in the roots of several plants, represents a renewable non-food biomass resource for the synthesis of bio-based products. Exploitation of inulin-containing feedstocks requires the integration of different processes, including inulinase production, saccharification of inulin, and microbial fermentation for the conversion of released sugars into added-value products. In this work paper, a new microbial source of inulinase, Penicillium lanosocoeruleum, was identified through the screening of a fungal library. Inulinase production using inulin as C-source was optimized, reaching up to 28 U mL–1 at the 4th day of growth. The fungal inulinase mixture (PlaI) was characterized for pH and temperature stability and activity profile, and its isoenzymes composition was investigated by proteomic strategies. Statistical optimization of inulin hydrolysis was performed using a central composite rotatable design (CCRD), by analyzing the effect of four factors. In the optimized conditions (T, 45.5°C; pH, 5.1; substrate concentration, 60 g L–1; enzyme loading, 50 U gsubstrate –1), up to 96% inulin is converted in fructose within 20 h. The integration of PlaI in a process for polyhydroxyalkanoate (PHA) production by Cupriavidus necator from inulin was tested in both separated hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF). A maximum of 3.2 g L–1 of PHB accumulation, corresponding to 82% polymer content, was achieved in the SSF. The proved efficiency in inulin hydrolysis and its effective integration into a SSF process pave the way to a profitable exploitation of the PlaI enzymatic mixture in inulin-based biorefineries.

Reversing the typical pH stability profile of the Trp-cage.

The Trp-cage, an 18-20 residue miniprotein, has emerged as a primary test system for evaluating computational fold prediction and folding rate determination efforts. As it turns out, a number of stabilizing interactions in the Trp-cage folded state have a strong pH dependence; all prior Trp-cage mutants have been destabilized under carboxylate-protonating conditions. Notable among the pH dependent stabilizing interactions within the Trp-cage are: (1) an Asp as the helix N-cap, (2) an H-bonded Asp9/Arg16 salt bridge, (3) an interaction between the chain termini which are in close spatial proximity, and (4) additional side chain interactions with Asp9. In the present study, we have prepared Trp-cage species that are significantly more stable at pH 2.5 (rather than 7) and quantitated the contribution of each interaction listed above. The Trp-cage structure remains constant with the pH change. The study has also provided measures of the stabilizing contribution of indole ring shielding from surface exposure and the destabilizing effects of an ionized Asp at the C-terminus of an α-helix.

Pharmaceutical Developability of Clinically Relevant Flavonoids like Curcumin: Why are We Failing?

Flavonoids are promising therapeutic class of polyphenolic biomolecules with the potential to alleviate many severe disease states with multi-factorial pathogenesis because of their inherent ability to regulate key biological cascades at the molecular, cellular and organism levels. However, these important polyphenolic compounds frequently pose drug delivery challenges to scientists and produce inefficient pharmacokineticpharmacodynamic activities. Importantly, despite their therapeutic potential and the focus of the scientists being on the formulation development front in the recent years, developing an ideal drug delivery system for some of the most important polyphenolic biomolecules such as curcumin is still a challenge. Hence, the basic intent of writing this article is to bring back the focus of the formulation scientists on one of the key physicochemical properties of these important biomolecules i.e., the importance of pH-stability profile of flavonoids, which could play vital role in the formulation developability aspects of important biomolecules such as curcumin by the selection of an ideal dosage form.

Partial purification and characterization of Cellulase-free xylanase production from bacillus licheniformis

A new isolate of Bacillus licheniformis SC produced extracellular xylanase when grown on 3% wheat bran, without xylanase multiplicity. The enzyme was partially purified by ammonium sulphate and gel filtration using Sephadex G-100 column. The purified xylanase showed optimum pH at 8.0 and half maximum activity at pH 4.5. The pH stability profile showed that it was more stable in the pH range of 6 to 8. Its activity increased with rise in temperature and maximum upto 60 0 C. The molecular mass for xylanase was 24.6 kDa respectively. The SDS-PAGE of the purified xylanase migrated as a single band. The Km was found to be 4.8 g/ml and the Vmax was 0.58 µmol/minute/ml for xylanase. In addition, the present xylanase was capable of producing high-quality xylo-oligosaccharides, indicating its application potential for biobleaching processes of pulp.

Inclusion complex of chrysin with sulfobutyl ether-β-cyclodextrin (Captisol®): Preparation, characterization, molecular modelling and in vitro anticancer activity

Present work demonstrates the impact of sulfobutyl ether-β-cyclodextrin (SBE-β-CD, Captisol®) complexation on the solubility, dissolution and pH stability of the poorly soluble polyphenol chrysin (CHR) by freeze-drying technique. Phase solubility indicated the 1:1 stoichiometry. FTIR, DSC, PXRD, and SEM analysis confirmed the formation of inclusion complex. Moreover, 1H NMR studies illustrated that the possible inclusion mode was through A-ring of the CHR which was further confirmed by molecular modelling. In addition, CHR/SBE-β-CD complex exhibited ∼108 times higher solubility and faster dissolution compared to CHR. The pH stability profile exhibited the protective effect of SBE-β-CD on the CHR at simulated pH. Also, in vitro cytotoxicity assay revealed an appreciable improvement in the anticancer activity of complex compared to free CHR, on MCF-7 and HeLa cell lines validating its superiority as a carrier.

Studies on the production of alkaline α-amylase from Bacillus subtilis CB-18.

Amylases are among the main enzymes used in food and other industries. They hydrolyse starch molecules into polymers composing glucose units. Amylases have potential applications in a number of industrial processes including foods and pharmaceutical industries. Alkaline α-amylase has the potential of hydrolysing starch under alkaline pH and is useful in the starch and textile industries and as an ingredient of detergents. Amylases are produced from plants, however, microbial production processes have dominated applications in the industries. Optimization of microbial production processes can result in improved enzyme yields. Amylase activity was assayed by incubating the enzyme solution (0.5 ml) with 1% soluble starch (0.5 ml) in 0.1 M Tris/HCl buffer (pH 8.5). After 30 minutes, the reaction was stopped by the addition of 4 mL of 3,5-dinitrosalicylic acid (DNS) reagent then heated for 10 min in boiling water bath and cooled in a refrigerator. Absorbance readings were used to estimate the units of enzyme activity from glucose standard curve. Hydrolysed native starches from cassava, rice, corn, coco yam, maize and potato and soluble starch were adjusted to pH 8.5 prior to incubation with crude enzyme solution. Reducing sugars produced were therefore determined. The effect of pH on enzyme activity of the alkaline α-amylase was determined by using buffer solutions of different pH (potassium phosphate buffer, 6.0-7.0; Tris-HCl buffer 7.5 to 9.0 and carbonate/bicarbonate buffer, pH 9.5-11) for enzyme assay. The pH stability profile of the enzyme was determined by incubating 0.5 ml of α-amylase enzyme in 0.1 M Tris/HCl buffer (pH 8.5) and 0.5 ml of 1% (w/v) soluble starch (Merck) in 0.1 M Tris/HCl buffer (pH 8.5) for 3 h in various buffers. The effect of temperature on enzyme activity was studied by incubating 0.5 mL of the enzyme solution contained in the test tube and 0.5 mL of 1% soluble starch (Merck) solution prepared in 0.1 M Tris/HCl buffer (pH 8.5) for 3 h at various temperatures (25, 30, 35, 40, 45, 50, 55 and 60°C) in a thermo static water bath. The reactions were stopped by adding DNS reagent. The enzyme activity was therefore determined. Thermal stability was studied by incubating 0.5 ml of enzyme solution in 0.1 M Tris/HCl buffer (pH 8.5) and 0.5 ml of 1% (w/v) soluble starch (Merck) in 0.1 M Tris/HCl buffer (pH 8.5) for 3 h at various temperatures (20, 30, 40, 50, 60 and 70°C) for 60 min. The enzyme displayed optimal activity at pH 8.0 at which it produced maximum specific activity of 34.3 units/mg protein. Maximum stability was at pH 8.0 to 9.0. Maximum activity was observed at temperature of 50°C while thermo stability of the enzyme was observed at 40-50°C. The enzyme displayed a wide range of activities on starch and caused the release of 5.86, 4.75, 5.98, 3.44, 3.96, 8.84 mg/mL reducing sugar from cassava, potato, cocoyam, corn, rice and soluble starch respectively. This investigation reports some biochemical characterization of alkaline α-amylase from Bacillus subtilis CB-18. The substrate specificities of this enzyme on various starches suggested that the alkaline α-amylase enzyme had combined activities on raw and soluble starch.

Surface acidity and solid-state compatibility of excipients with an acid-sensitive API: case study of atorvastatin calcium.

The objectives of this study were to measure the apparent surface acidity of common excipients and to correlate the acidity with the chemical stability of an acid-sensitive active pharmaceutical ingredient (API) in binary API-excipient powder mixtures. The acidity of 26 solid excipients was determined by two methods, (i) by measuring the pH of their suspensions or solutions and (ii) the pH equivalent (pHeq) measured via ionization of probe molecules deposited on the surface of the excipients. The chemical stability of an API, atorvastatin calcium (AC), in mixtures with the excipients was evaluated by monitoring the appearance of an acid-induced degradant, atorvastatin lactone, under accelerated storage conditions. The extent of lactone formation in AC-excipient mixtures was presented as a function of either solution/suspension pH or pHeq. No lactone formation was observed in mixtures with excipients having pHeq > 6, while the lactone levels were pronounced (> 0.6% after 6 weeks at 50°C/20% RH) with excipients exhibiting pHeq < 3. The three pHeq regions (> 6, 3-6, and < 3) were consistent with the reported solution pH-stability profile of AC. In contrast to the pHeq scale, lactone formation did not show any clear trend when plotted as a function of the suspension/solution pH. Two mechanisms to explain the discrepancy between the suspension/solution pH and the chemical stability data were discussed. Acidic excipients, which are expected to be incompatible with an acid-sensitive API, were identified based on pHeq measurements. The incompatibility prediction was confirmed in the chemical stability tests using AC as an example of an acid-sensitive API.

Pre-formulation studies of resveratrol

Context: Resveratrol, a natural compound found in grapes, has potential chemotherapy effects but very low oral bioavailability in humans.Objective: To evaluate the solubility, pH stability profile, plasma protein binding (PPB) and stability in plasma for resveratrol.Methods: Solubility of resveratrol was measured in 10 common solvents at 25 °C using HPLC. The solution state pH stability of resveratrol was assessed in various United States Pharmacopeia buffers ranging from pH 2 to 10 for 24 h at 37 °C. Samples were analyzed up to 24 h. Human PPB was determined using ultracentrifugation technique. Standard solutions of drug were spiked to blank human plasma to yield final concentrations of 5, 12.5 or 25 μg/mL for determination. Finally, stability of resveratrol in human and rat plasma was also assessed at 37 °C. Aliquots of blank plasma were spiked with a standard drug concentration to yield final plasma concentration of 50 μg/mL. Samples were analyzed for resveratrol concentration up to 96 h.Results: Resveratrol has wide solubility ranging from 0.05 mg/mL in water to 374 mg/mL in polyethylene glycol 400 (PEG-400). Resveratrol is relatively stable above pH 6 and has maximum degradation at pH 9. The mean PPB of resveratrol is 98.3%. Resveratrol degrades in human and rat plasma in a first-order process with mean half lives of 54 and 25 h, respectively.Conclusion: Resveratrol is more soluble in alcohol and PEG-400 and stable in acidic pH. It binds highly to plasma proteins and degrades slower in human then rat plasma.

Development of a recombinant fusion protein vaccine formulation to protect against Streptococcus pyogenes

Diseases resulting from infection by group A streptococcus (GAS) are an increasing burden on global health. A novel vaccine was developed targeting infection by Streptococcus pyogenes. The vaccine incorporates a recombinant fusion protein antigen (SpeAB) which was engineered by combining inactive mutant forms of streptococcal pyrogenic exotoxin A (SpeA) and streptococcal pyrogenic exotoxin B (SpeB) from S. pyogenes. A rational, scientific approach to vaccine development was utilized to determine optimal formulation conditions with aluminum adjuvants. Investigations of the pH stability profile of SpeAB concluded the antigen was most stable near pH 8. Incorporation of the stabilizers sucrose and mannitol significantly enhanced the stability of the antigen. Vaccines were formulated in which most of the SpeAB was adsorbed to the adjuvant or remained in solution. A SpeAB vaccine formulation, stabilized with sucrose, in which the antigen remains adsorbed to the aluminum adjuvant retained the greatest potency as determined by evaluation of neutralizing antibody responses in mice. This vaccine has great potential to provide a safe and effective method for prevention of GAS disease.

Effect of storage conditions on the stability of β-lapachone in solid state and in solution

In this work, the effects of several technological factors on the stability of β-lapachone (βLAP) in solution and in the solid state were investigated. The effects of relative humidity and light on the stability of βLAP in the solid state were studied. Samples were characterized by liquid chromatography, thermal analysis, X-ray powder diffraction and optical microscopy. In solution, the effects of light conditions and additives (cyclodextrins) were also evaluated. Molecular modelling was used to support the degradation mechanism involved. Additionally, the pH stability profile of βLAP was established. The synergism of relative humidity and light promoted degradation of βLAP in the solid state, with important consequences for the physical and chemical characteristics of the drug after storage. Random methyl-β-cyclodextrin was able to protect the drug against the hydrolytic process in darkness. However, it accelerated the drug decomposition by photolysis in light conditions. According to the pH stability profile, βLAP undergoes an alkaline hydrolysis, its maximum stability pH being over the range 2-4. These studies provide useful information regarding the optimal storage conditions and formulations of βLAP.

Degradation of BMS-753493, a novel epothilone folate conjugate anticancer agent

BMS-753493 is a folate-targeted candidate being developed for the treatment of cancer. As part of preformulation efforts, our aim was twofold – to understand the major degradation pathways and, study its kinetics of degradation to aid drug product development. Given the complexity of degradation, BMS-748285, the epothilone moiety of BMS-753493 was used as model compound to evaluate the major degradation pathway viz; macrolactone versus aziridine ring hydrolysis. Hydrolysis of BMS-753493 was studied in the pH range of 1.5–9.4 in 0.05 M buffers at 0.5 ionic strength and 5–40°C. Three major pathways were identified; carbonate ester hydrolysis and hydrolysis of aziridine and macrolactone rings resulting in addition products with identical masses (m/z = 794) in the pH range of 5–7.5. Similarly, two addition products, D1 and D2 (m/z = 555) were also formed on hydrolysis of BMS-748285 under neutral pH conditions. The reaction products from BMS-748285 were isolated and characterized using LC-MS and LC-SPE-NMR (1-D 1H and 2-D HMBC, heteronuclear single quantum coherence) analyses. LC-NMR analysis indicated an intact aziridine ring and opened macrolactone ring, resulting in D1 and D2, an isomeric hydroxy acid pair resulting from an alkyl oxygen cleavage. By analogy to BMS-748285, BMS-753493 was also postulated to undergo alkyl cleavage of the macrolactone, forming two epimeric hydroxy acids under neutral pH. The pH-stability data were also consistent with these findings. Additionally, the degradation kinetics for BMS-753493, indicated a U-shaped pH-stability profile with maximum stability at pH 7. Based on the stability and solubility considerations, the pH range of 6–7 was optimal for an injectible drug product development.

Determination of Solid-State Acidity of Chitin–Metal Silicates and their Effect on the Degradation of Cephalosporin Antibiotics

It was of interest to determine the solid-state acidity of chitin-metal silicate coprocessed excipients and to correlate this acidity to the chemical stability of cefotaxime sodium in the presence of the aforementioned excipients. The solid-state acidities of chitin aluminum silicate, chitin magnesium silicate, and chitin calcium silicate were determined by reflectance spectroscopy using structurally different dye molecules. The chemical stability of cefotaxime sodium was assessed at 50 °C in a 4% (w/v) slurry system in the pH range 6.6-10.5 and in the solid-state in the Hammett acidity range 6.1-7.8. The solid-state acidity was found to be reproducible because one or more structurally different dye molecules gave reliable solid-state acidity values. A significant discrepancy in pH stability profile of cefotaxime sodium between the solid-state and the slurry system was observed. Furthermore, chitin aluminum silicate showed minimum drug stability in the solid-state, close to where the maximum drug stability in the slurry was observed. This unexpected effect might be ascribed to the catalytic properties of chitin aluminum silicate. The slurry method was not able to predict efficiently the solid-state surface acidity and stability of cefotaxime sodium. Moreover, the solid-state chemical stability might be influenced by factors other than the solid-state acidity.

Clotrimazole nanoemulsion for malaria chemotherapy. Part I: Preformulation studies, formulation design and physicochemical evaluation

Clotrimazole was formulated in nanoemulsion based system with the aim of improving its solubility and dissolution, which can further used for its preclinical evaluation. Clotrimazole nanoemulsion was prepared using spontaneous nanoemulsification method. Preformulation studies were preformed to evaluate drug-excipient compatibility, solution state pH stability and pH solubility profile. Solubility of clotrimazole in oils, surfactants and cosurfactants was determined to identify nanoemulsion components. Surfactants and cosurfactants were screened for their ability to emulsify selected oily phases. Phase diagrams were constructed to identify area of nanoemulsification. Influence of clotrimazole and pH of dilution medium on phase behavior were assessed. Drug-excipient chemical compatibility study facilitated to anticipate acid catalyzed degradation of clotrimazole. The pH of nanoemulsion was adjusted to 7.5, which could stabilize clotrimazole. Nanoemulsion composed of Capryol 90, Solutol HS 15 and Gelucire 44/14 enhanced solubility of clotrimazole up to 25mg/ml. The optimized clotrimazole nanoemulsion could withstand the extensive dilution and did not show any phase separation or drug precipitation. The nanoemulsion exhibited mean globule size <25nm, which was not affected by pH of dilution medium. Dissolution profile of clotrimazole nanoemulsion in various media showed 100% drug release within 15min irrespective of pH of medium.

The native conformation of plasmepsin II is kinetically trapped at neutral pH

Plasmepsin II (PMII), an aspartic protease from the malarial parasite Plasmodium falciparum, represents a model for understanding protease structure/function relationships due to its unique structure and properties. The present study undertook a thermodynamic and kinetic analysis of the PMII folding mechanism and a pH stability profile. Differential scanning calorimetry revealed that the native state of PMII (Np) was irreversibly unfolded, and in the pH range of 6.5–8.0, PMII refolds to a denatured state (Rp) with higher thermal stability than Np. Rp could also be formed upon partially unfolding PMII at pH 11.0 and 37°C for 2h, followed by adjustment to a pH in the range of 6.5–8.0. While Rp could be folded/unfolded reversibly, Np was shown to exist as a kinetically trapped state. By examining the unfolding kinetics of Np and the kinetics of Rp folding to Np at 25°C, it was found that Np is kinetically trapped by an unfolding barrier of 25.5kcal/mol, and yet once unfolded, is prevented from folding by a comparable folding barrier. The folding mechanism of PMII is similar to that reported for pepsin. It is hypothesized that the PMII zymogen also utilizes a prosegment-catalyzed folding mechanism.

Characterization of purified Polyphenol oxidase (PPO) from (Lactuca serriola L.) Prickly lettuce

In this study, purified PPO from Prickly lettuce were obratind from a previous characterization study. The results showed that the optimum pH were 6.5 and the pH stability profile showed that the enzyme were more stable at pH values 5.5-8, the optimum temperature for enzyme activity were 35ºC and the enzyme were stable at 35ºC for 10 min. the activation energy for conversion of substrate to product was 39.7194 Kj/mol, the purified enzyme lost its activity after 5 days of storage at 25ºC, while it lost (73 , 16)% of primary activity after 21 days of storage at 4 and -18ºC respectively, the purified enzyme contains 7.2% carbohydrates. It was noticed that all treated of enzyme with L-Cystein, Sodium metabisulphate and Dithiothretol at 1 and 10 mM revealed high inhibition for the enzyme activity, followed by citric acid, L-Ascorbic acid, EDTA and Potassium cyanide, while both Thiourea and Sodium benzoate showed less inhibitory effect. The Km and Vmax were (4.762 , 0.0588) mM/min respectively, while the coefficient Vmax/Km were 0.01235 min-1 for the purified enzyme when used Catechol as a substrate.

NP-647, a novel TRH analogue: Investigating physicochemical parameters critical for its oral and parenteral delivery

NP-647 ( l-pGlu-(2-propyl)- l-His- l-ProNH 2) is a novel thyrotropin releasing hormone (TRH) analogue, with potential antiepileptic activity. In the present study, the physicochemical parameters of NP-647, including its solid state properties, dissociation constant, partition coefficient, solubility (intrinsic solubility and pH-solubility profile) and stability (gastrointestinal enzymatic stability, pH-stability profile and temperature stability) were investigated for their criticality for oral and parenteral delivery. NP-647 was characterized as amorphous material having glass transition temperature of 66.73 °C at 50% RH. It was found very hygroscopic with deliquescent in nature. p K a of the compound, as determined using potentiometric titration, was found to be 7.2 ± 0.02 (basic). Intrinsic solubility and pH-solubility behavior were determined using dissolution titration template method. NP-647 has intrinsic solubility of 2.4 ± 0.01 mg mL −1. Partition/distribution studies indicate that NP-647 has a low log P (−1.07 ± 0.06) and log D 7.4 (−1.20 ± 0.02), characteristic of hydrophilic molecule. It was found most stable in tartrate buffer of pH of 5.0. Arrhenius plot of NP-647 suggest its half life of ∼3.2 years and shelf life of ∼6 months. These studies conclude that amorphous nature of NP-647 with deliquescent property will be critical in its solid oral dosage formulation and need to be investigated further.

Solid-State Surface Acidity and pH-Stability Profiles of Amorphous Quinapril Hydrochloride and Silicate Formulations

To determine the surface acidity and stability profiles of quinapril hydrochloride (QHCl) coground with silicates, solid-state equivalent pH (pHeq) of amorphous samples was measured by diffuse reflectance spectroscopy using pH indicator probes. Calibration curves for pH indicators were developed in buffer solutions. Amorphous samples with and without pH indicators were prepared by cryo-grinding. Different pH grades of silicates and various QHCl/silicate ratios were used to make amorphous samples over a range of surface acidity. Diffuse reflectance spectra of amorphous samples were measured and pHeq was determined using the calibration curves of pH indicators developed in solution. Suspension pH of amorphous samples was also measured. The chemical stability of coground amorphous samples was assessed at 40 degrees C and 0% or 48% RH. The chemical stability of neat amorphous quinapril lyophilized from solutions over a range of pH was also assessed at 40 degrees C/0% RH and the reconstituted pH-stability profile of lyophiles was determined. For all silicate and QHCl/silicate amorphous samples, the same pH rank order was obtained based on pHeq and suspension pH. However, the pHeq was significantly lower than the corresponding suspension pH. Discrepancies between pH-stability profiles based on the pHeq and the suspension pH were observed. In general, the pHeq- and reconstituted pH-stability profiles were essentially identical, but the suspension pH-stability profile deviated from the reconstituted pH-stability profile by 2-3 pH units. The results indicate that solid-state surface acidity measurement provides a more accurate prediction of the effective surface acidity of amorphous formulations than the suspension pH. In conclusion, solid-state surface acidity measurement of excipients and solid formulations using pH indicator probes as surrogates can be used to determine the ionization state of the drug and to predict the chemical stability profile of the drug in actual solid formulations.

PH-Stability and Thermal Properties of Microbial Transglutaminase-Treated Whey Protein Isolate

Whey protein isolate (WPI) was treated to various extents using microbial transglutaminase (MTGase) and changes in pH-stability and thermal stability of its protein components were investigated. The MTGase treatment significantly increased the denaturation temperature (T(d)) of beta-lactoglobulin in WPI, from 71.84 degrees C in the untreated sample to 78.50 degrees C after 30 h of incubation with MTGase. The enthalpy change of denaturation of WPI did not change upon cross-linking, indicating that the increase in T(d) was primarily due to covalent cross-linking and not due to an increase in nonpolar interactions within the protein. The surface hydrophobicity (S(o)) of the protein decreased upon cross-linking; however, this decrease was not due to burial of the surface hydrophobic cavities in the protein interior, but due to occlusion of the hydrophobic cavities to the fluorescent probes. Fluorescence emission and circular dichroism spectroscopic analyses revealed no major changes in the secondary and tertiary conformations as a result of cross-linking. However, unlike native WPI, the cross-linked protein exhibited a U-shaped pH-stability profile with maximum turbidity at pH 4.0-4.5. The study revealed that even though enzymatic cross-linking significantly improved the T(d) of beta-lactoglobulin in WPI without causing major structural changes, a reduction in the hydrophilic-hydrophobic balance of the protein surface as a result of elimination of the positive charge on lysyl residues caused precipitation at pH 4.0-4.5.