Quantification and Characterization of Subvisible Proteinaceous Particles in Opalescent mAb Formulations Using Micro-Flow Imaging

Micro-flow imaging (MFItm) has been shown to be more sensitive than light obscuration (LO) methods for measuring subvisible proteinaceous particles in protein formulations. Given the potential challenges in detecting particulates in opalescent mAb formulations, the accuracy of MFI to size and count particles in opalescent solutions was investigated and compared to LO and membrane microscopy methods. Proteinaceous monoclonal antibody (mAb) particles, generated either by chemical denaturation or agitation stress, polystyrene and glass particles were used as model systems for measurements in opalescent mAb solutions. The sizing and counting accuracies of MFI were unaffected by the opalescence of the medium. Using glass particles as a model system for proteinaceous particles, MFI was able to detect relatively low particle concentrations (~ 10/mL) in opalescent solutions. MFI showed excellent linearity (R2 = 0.9969) for quantifying proteinaceous particles in opalescent solutions over a wide range of particle concentrations (~ 20-160,000/mL). Analyses of MFI particle image intensities revealed significant differences in the transparency of proteinaceous particles as a function of their size and mode of generation. LO method significantly underestimated proteinaceous particles, particularly those in the 2–10 μm size range. The less opaque proteinaceous particles were relatively more underestimated by the LO method in opalescent solutions.     

Using a Model-based Material Sparing Approach for Formulation and Process Development of a Roller Compacted Drug Product

Pharmaceutical Research - The present work details a material sparing approach that combines material profiling with Instron uniaxial die-punch tester and use of a roller compaction mathematical...     

Spectral Markers for T Cell Death and Apoptosis—A Pilot Study on Cell Therapy Drug Product Characterization Using Raman Spectroscopy

Application of Raman spectroscopy as a T cell characterization tool supporting cell therapy drug product development has been evaluated. Statistically significant correlations between a set of Raman signals and established flow cytometry markers associated with apoptosis of T cells detected during drug product cryopreservation are presented in this study. Our study results demonstrate the potential of Raman spectroscopy for label-free measurements of T cell characteristics relevant to cell therapy product design and process control.     

Residue-Specific Impact of EDTA and Methionine on Protein Oxidation in Biotherapeutics Formulations Using an Integrated Biotherapeutics Drug Product Development Workflow

The rational design and selection of formulation composition to meet molecule-specific and product-specific needs are critical for biotherapeutics development to ensure physical and chemical stability. This work, based on three antibody-based (mAb) proteins (mAbA, mAbB, and mAbC), evaluates residue-specific impact of EDTA and methionine on protein oxidation, using an integrated biotherapeutics drug product development workflow. This workflow includes statistical experimental design, high-throughput experimental automation and execution, structure-based in silico modeling, inferential statistical analysis, and enhanced interactive data visualization of large datasets. This oxidation study evaluates the impact of formulation parameters including pH, protein concentration, and the presence of polysorbate 80 on the oxidation of specific conserved and variable residues of mAbs A, B, and C in the presence of stressors (iron, peroxide) and/or protectants (EDTA, L-methionine). Residue-specific analysis by automated high-throughput peptide mapping demonstrates differential residue-specific effects of EDTA and methionine in protecting against oxidation, highlighting the need for molecule-specific and product-specific selection of these excipients during formulation development. Computational modeling based on a homology model and the two-shell water coordination method (WCN) was employed to gain mechanistic understanding of residue-specific oxidation susceptibility of methionine residues. The computational determinants of local solvent exposure of methionine residues showed good correlation of WCN with experimentally determined oxidation for corresponding residues. The rapid generation of high-resolution data, statistical data analysis and interactive visualization of the high-throughput residue-level data containing ～200 unique formulations facilitate residue-specific, molecule-specific and product-specific oxidation (global and local) assessment for oxidation protectants during early development for mAbs and related mAb-based modalities.     

Characterization of a Synthetic Anionic Vector for Oligonucleotide Delivery Using in Vivo Whole Body Dynamic Imaging

Purpose. To compare the pharmacokinetics and bioavailability of an oligonucleotide delivered in a free form or using cationic or anionic synthetic carrier systems. Methods. Whole body dynamic quantitative imaging and metabolism of a HIV antisense oligonucleotide intravenously administered either free or incorporated into synthetic carriers were compared in baboons, using non invasive positron emission tomography and an enzyme-based competitive hybridization assay, respectively. Results. In its free form, the oligonucleotide showed high liver and kidney concentration, rapid plasmatic degradation and elimination from the body. Use of a cationic vector slightly protected the oligonucleotide against degradation and enhanced uptake by the reticulo-endothelial system. In contrast, the anionic vector dramatically enhanced the uptake in several organs, including the lungs, spleen and brain, with a prolonged accumulation of radioactivity in the brain. Using this vector, intact oligonucleotide was detected in plasma for up to two hours after injection, and the T_1/2 and distribution volume increased by 4- and 7-fold, respectively. No evidence of toxicity was found after a single dose administration. Conclusions. The anionic vector improves significantly the bioavailability and the pharmacokinetics of the oligonucleotide, and is a promising delivery system for in vivo administration of therapeutic nucleic acids.     

Stress Factors in Primary Packaging,Transportation and Handling of Protein Drug Products and Their Impact on Product Quality

Protein-based biologic drugs encounter a variety of stress factors during drug substance (DS) and drug product (DP) manufacturing, and the subsequent steps that result in clinical administration by the end user. This article is the third in a series of commentaries on these stress factors and their effects on biotherapeutics. It focuses on assessing the potential negative impact from primary packaging, transportation, and handling on the quality of the DP. The risk factors include ingress of hazardous materials such as oxidizing residuals from the sterilization process, delamination- or rubber stopper-derived particles, silicone oil droplets, and leachables into the formulation, as well as surface interactions between the protein and packaging materials, all of which may cause protein degradation. The type of primary packaging container used (such as vials and prefilled syringes) may substantially influence the impact of transportation and handling stresses on DP Critical Quality Attributes (CQAs). Mitigations via process development and robustness studies as well as control strategies for DP CQAs are discussed, along with current industry best practices for scale-down and in-use stability studies. We conclude that more research is needed on postproduction transportation and handling practices and their implications for protein DP quality.     

Fluorescence Single Particle Tracking for the Characterization of Submicron Protein Aggregates in Biological Fluids and Complex Formulations

Purpose

To evaluate the potential of fluorescence single particle tracking (fSPT) for the characterization of submicron protein aggregates in human serum, plasma and formulations containing human serum albumin (HSA).

Methods

A monoclonal IgG was covalently labeled with a fluorescent dye and cross-linked with glutaraldehyde. IgG aggregates and fluorescent beads of 0.1 ??m (control) were diluted in buffer, serum and plasma, and their size distributions were analyzed by fSPT and nanoparticle tracking analysis (NTA). In a separate experiment, IgG and HSA, fluorescently labeled with different dyes, were mixed and subjected to heat stress. The stressed sample was analyzed by fSPT using a dual color mode and by NTA.

Results

The accuracy and precision of fSPT proved to be comparable to NTA. fSPT was able to successfully measure all the samples in buffer, serum and plasma. The average size of the cross-linked protein aggregates showed a slight increase in biological fluids. Moreover, fSPT analysis showed that a significant proportion of the aggregates formed by subjecting an IgG/HSA mixture to heat stress were composed of both proteins.

Conclusion

fSPT is a powerful technique for the characterization of submicron protein aggregates in biological fluids and complex formulations.     

Detecting Crystallinity in Amorphous Solid Dispersions Using Dissolution Testing: Considerations on Properties of Drug Substance,Drug Product,and Selection of Dissolution Media

Dissolution testing has long been used to monitor product quality. Its role in quality control of amorphous solid dispersion (ASD) formulations is relatively new. In the presence of the crystalline phase, the dissolution of ASDs is determined by the dynamics between the dissolution rate of the amorphous solids and the rate of crystal growth. The detection of crystalline phase by dissolution test has not been well understood in the context of drug properties, formulation characteristics and dissolution test variables. This study systematically evaluated the impact of key parameters such as intrinsic crystallization tendency of the API, drug loading, extent of dissolution sink conditions and level of crystallinity on the ASD dissolution behavior. The results indicated diverse dissolution behaviors due to the differences in the intrinsic crystallization propensity of the drug, the drug loading, the ASD polymers and the dissolution sink index. Each of the complex dissolution profiles were interpreted based on visual observations during dissolution, the appropriate sink index based on the amorphous solubility, and the competition between drug dissolution versus crystallization. The findings of this study provide insights towards the various considerations that should be taken into account towards rationally developing a discriminatory dissolution method.     

Predicting Formulation Conditions During Ultrafiltration and Dilution to Drug Substance Using a Donnan Model with Homology-Model Based Protein Charge

In the manufacturing of therapeutic monoclonal antibodies (mAbs), the final steps of the purification process are typically ultrafiltration/diafiltration (UF/DF), dilution, and conditioning. These steps are developed such that the final drug substance (DS) is formulated to the desired mAb, buffer, and excipient concentrations. To develop these processes, process and formulation development scientists often perform experiments to account for the Gibbs-Donnan and volume-exclusion effects during UF/DF, which affect the output pH and buffer concentration of the UF/DF process. This work describes the development of an in silico model for predicting the DS pH and buffer concentration after accounting for the Gibbs-Donnan and volume-exclusion effects during the UF/DF operation and the subsequent dilution and conditioning steps. The model was validated using statistical analysis to compare model predictions against experimental results for nine molecules of varying protein concentrations and formulations. In addition, our results showed that the structure-based in silico approach used to calculate the protein charge was more accurate than a sequence-based approach. Finally, we used the model to gain fundamental insights about the Gibbs-Donnan effect by highlighting the role of the protein charge concentration (the protein concentration multiplied with protein charge at the formulation pH) on the Gibbs-Donnan effect. Overall, this work demonstrates that the Gibbs-Donnan and volume-exclusions effects can be predicted using an in silico model, potentially alleviating the need for experiments.     

Characterization and Compatibility Studies between Policosanol,a New Hypocholesterolemic Drug,and Tablet Excipients Using Differential Scanning Calorimetry (DSC)

Characterisation of policosanol, a new active principle composed of 8 high molecular weight fatty alcohols, viz. 1-tetracosanol, 1-hexacosanol, 1-heptacosanol, 1-octacosanol, 1-nonacosanol, 1-triacontanol, 1-dotriacontanol, and 1-tetratriacontanol, shows it have a very stable, well defined composition which is reproducible from batch to batch. Compatibility studies by differential scanning calorimetry (DSC) and thermogravimetry (TG) gave very useful physicochemical information and reveal the characteristic transitions, as well as the thermal stability of this drug. DSC facilitated compatibility studies between policosanol and several tablet excipients generally used for the manufacture of this pharmaceutical dosage form. It is seen that the combination of policosanol with each excipient in every one of the ratios used did not produce any changes in the melting point of policosanol or those of the excipients mixed with it. Also, no new peaks were observed in the policosanol/excipient mixtures. It can be concluded from these results that no dissolution of policosanol in the excipients occurs, and also that no physicochemical interactions take place between them.     

Identification of an Adduct Impurity of an Active Pharmaceutical Ingredient and a Leachable in an Ophthalmic Drug Product Using LC-QTOF

Impurity investigations are important in pharmaceutical development to ensure drug purity and safety for the patient. The impurities typically found in drug products are degradants or reaction products of the active pharmaceutical ingredient (API) or leachable compounds from the container closure system. However, secondary reactions may also occur between API degradants, excipient impurities, residual solvents, and leachables to form adduct impurities. We hereby report an adduct-forming interaction of API (moxifloxacin) with a leachable compound (ethylene glycol monoformate) in moxifloxacin ophthalmic solution. The leachable compound originated from a low-density polyethylene bottle used in the packaging of drug products. The adduct impurity was tentatively identified as 1-cyclopropyl-6-fluoro-7-(1-(2-(formyloxy)ethyl) octahydro-6H-pyrrolo[3,4-b]pyridin-6-yl)-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (C₂₄H₂₈FN₃O₆, MW = 473.19621) using accurate mass LC-QTOF analysis. The mass accuracy error between the theoretical mass and the experimental mass of an impurity was found to be 0.2 ppm. An MS/MS analysis was utilized to provide mass spectrometry fragments to support verification of the proposed structure.     

A Theoretical Basis for a Biopharmaceutic Drug Classification: The Correlation of in Vitro Drug Product Dissolution and in Vivo Bioavailability

A biopharmaceutics drug classification scheme for correlating in vitro drug product dissolution and in vivo bioavailability is proposed based on recognizing that drug dissolution and gastrointestinal permeability are the fundamental parameters controlling rate and extent of drug absorption. This analysis uses a transport model and human permeability results for estimating in vivo drug absorption to illustrate the primary importance of solubility and permeability on drug absorption. The fundamental parameters which define oral drug absorption in humans resulting from this analysis are discussed and used as a basis for this classification scheme. These Biopharmaceutic Drug Classes are defined as: Case 1. High solubility-high permeability drugs, Case 2. Low solubility-high permeability drugs, Case 3. High solubility-low permeability drugs, and Case 4. Low solubility-low permeability drugs. Based on this classification scheme, suggestions are made for setting standards for in vitro drug dissolution testing methodology which will correlate with the in vivo process. This methodology must be based on the physiological and physical chemical properties controlling drug absorption. This analysis points out conditions under which no in vitro-in vivo correlation may be expected e.g. rapidly dissolving low permeability drugs. Furthermore, it is suggested for example that for very rapidly dissolving high solubility drugs, e.g. 85% dissolution in less than 15 minutes, a simple one point dissolution test, is all that may be needed to insure bioavailability. For slowly dissolving drugs a dissolution profile is required with multiple time points in systems which would include low pH, physiological pH, and surfactants and the in vitro conditions should mimic the in vivo processes. This classification scheme provides a basis for establishing in vitro-in vivo correlations and for estimating the absorption of drugs based on the fundamental dissolution and permeability properties of physiologic importance.     

金针菇中具抑瘤活性的碱性蛋白火菇素的分离纯化和性质研究

目的对一种具有抑瘤活性的碱性蛋白火菇素进行分离纯化和性质研究。方法应用硫酸铵、乙醇分级沉淀，柱色谱等方法进行分离纯化。电泳分析、氨基酸组成分析、质谱分析等方法被用于火菇素的理化性质研究。结果火菇素被提纯至电泳纯并结晶。经性质研究确定火菇素的分子量为19891.13Da,pI=8.9,λmax=276-278nm,λmin=250nm，并缺少甲硫氨酸。通过基质辅助激光解吸离子化质谱（MALDI-MS）分析，测得了火菇素的指纹图谱，表明它为一种新蛋白。结论火菇素被首次提纯至电泳纯，并首次叙述了它的理化性质。     

Synthesis,Pharmacology, Crystal Properties,and Quantitative Solvation Studies from a Drug Transport Perspective for Three New 1,2,4-thiadiazoles

A novel 1,2,4-thiadiazoles were synthesized. Crystal structures of these compounds were solved by X-ray diffraction experiments and comparative analysis of molecular conformational states, packing architecture, and hydrogen bonds networks were carried out. Thermodynamic aspects of sublimation processes of studied compounds were determined using temperature dependencies of vapor pressure. Thermophysical characteristics of the molecular crystals were obtained and compared with the sublimation and structural parameters. Solubility and solvation processes of 1,2,4-thiadiazoles in buffer, n-hexane and n-octanol were studied within the wide range of temperature intervals and thermodynamic functions were calculated. Specific and nonspecific interactions of molecules resolved in crystals and solvents were estimated and compared. Distribution processes of compounds in buffer/n-octanol and buffer/n-hexane systems (describing different types of membranes) were investigated. Analysis of transfer processes of studied molecules from the buffer to n-octanol/n-hexane phases was carried out by the diagram method with evaluation of the enthalpic and entropic terms. This approach allows us to design drug molecules with optimal passive transport properties. Calcium-blocking properties of the substances were evaluated. © 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:3754-3768, 2010     

A Rapid, Stability-Indicating RP-HPLC Method for the Simultaneous Determination of Formoterol Fumarate, Tiotropium Bromide, and Ciclesonide in a Pulmonary Drug Product

A stability-indicating reversed-phase high performance liquid chromatography (RP-HPLC) method was developed for the simultaneous determination of Formoterol fumarate (FOR), Tiotropium bromide (TRI), and Ciclesonide (CLS) in a pulmonary drug product. The desired chromatographic separation was achieved on the Zorbax SB C8, 5 μm (150 × 4.6 mm) column, using gradient elution at 230 nm detector wavelength. The optimized mobile phase consisted of a 0.2 % v/v perchloric acid as solvent-A and acetonitrile as solvent-B. The developed method separated FOR, TRI, and CLS in the presence of its five unknown degradation products within 10 minutes. The stability-indicating capability was established by forced degradation experiments and the separation of unknown degradation products. The developed RP-HPLC method was validated according to the International Conference on Harmonization (ICH) guidelines. This validated method was applied for the simultaneous estimation of FOR, TRI, and CLS in commercially available Triohale^® pMDI (Pressurized Metered-Dose Inhaler) samples. Furthermore, this method can be extended for individual estimation of FOR, TRI, and CLS in various commercially available pulmonary dosage forms.     

Synthesis,Characterization, and Crystal Chemistry of Tasimelteon,a Melatonin Agonist,in Its Anhydrous and Hemihydrate Forms

Two crystalline forms of tasimelteon, a drug approved by the U.S. Food and Drug Administration for the treatment of non-24-h sleep-wake disorder, have been studied by single crystal and powder diffraction analyses, thermogravimetric analysis, differential scanning calorimetry, spectroscopic, and optical methods. The synthetic method forming tasimelteon is described in detail, with its full analytical, spectroscopic, and enantiopurity characterization. Solid tasimelteon hemihydrate, C₁₅H₁₉NO₂·0.5H₂O, is tetragonal with a = b = 7.3573(2) Å, c = 52.062(2) Å, V = 2818.1(2) ų; Z = 8. Its crystal structure has been solved and refined in the P4₃2₁2 space group, showing the occurrence of polymeric (H-bonded) slabs, thanks to the presence of water molecule (O_W) tetrahedrally linked to 4 distinct tasimelteon molecules in a N₂(O_W)O₂ fashion. The anhydrous form of tasimelteon, C₁₅H₁₉NO₂, crystallizes in the monoclinic P2₁ space group, with a = 11.130(4), b = 4.907(2), c = 12.230(6) Å, β = 91.03(3)°, V = 667.8(5) ų; Z = 2. Thanks to the availability of good-quality specimens, the structure of the latter phase was solved by conventional single-crystal diffraction analysis, showing short intermolecular C=O^…H–N interactions between (translationally related) tasimelteon molecules, forming, in the crystal, well-defined chains running along the b axis. The morphology of the 2 crystal forms has been analyzed by the means of optical microscopy and particle size distribution analysis. Worthy of note, the newly determined crystal structures enable the successful usage of full-pattern matching X-ray-based quantitative analyses of batches of industrial interest, in search for contamination or phase stability issues.     

Using Hair Analysis,Urinalysis, and Self-Reports to Estimate Drug Use in a Sample of Detained Juveniles

This paper reports select findings of a research project designed to estimate drug use prevalence in a youthful offender population using hair analysis as well as urine testing and interviewing. The project was carried out in Cleveland, Ohio, and Pinellas County, Florida. The findings are consistent with earlier reports on prevalence estimations utilizing a bioassay component. Generally, respondents report drug use infrequently and test positive by assays at rates greater than self-reported use. Urinalysis indicates more drug prevalence than does interview. Hair assays, which have a greater retrospective time window, show even more prevalence than does urine testing. The project affirms results reported in 1994b Feucht, Stephens, and Walker. [Translations are provided in the International Abstracts Section of this issue.]     

Polymeric Nanoparticles for Increased Oral Bioavailability and Rapid Absorption Using Celecoxib as a Model of a Low-Solubility, High-Permeability Drug

Purpose  

To demonstrate drug/polymer nanoparticles can increase the rate and extent of oral absorption of a low-solubility, high-permeability drug.