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Journal of Pharmaceutical Sciences
Journal Prestige (SJR): 0.984
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  Hybrid Journal Hybrid journal (It can contain Open Access articles)
ISSN (Print) 0022-3549 - ISSN (Online) 1520-6017
Published by Elsevier Homepage  [3161 journals]
  • Feasibility Study for the Rectal Route of Administration for Gentamicin
           Evaluated in the Neonatal Minipig Model
    • Abstract: Publication date: February 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 2Author(s): David H. McAdams, Manjari Lal, Manshun Lai, Manjari Quintanar-SolaresAbstractNeonatal infections are a major cause of newborn mortality in low- and middle-income countries, particularly in areas without access to inpatient care. To address this, the World Health Organization developed guidelines for delivering simplified antibiotic regimens (oral amoxicillin and intramuscular gentamicin) in outpatient settings to young infants with suspected serious bacterial infection when referral is not feasible. However, there are still limitations to access, as the regimen requires a health care provider trained in giving intramuscular injections to infants. To provide a needle-free, simplified alternate to intramuscular delivery, PATH investigated the feasibility of the rectal administration of gentamicin. Potential formulations were screened by in vitro testing, and 2 liquid enema formulations and a cocoa butter suppository were developed and evaluated in a preclinical study of the rectal uptake of gentamicin in a neonatal minipig model. Sera samples from the control group, dosed by intramuscular injection, resulted in expected sera concentrations of gentamicin, but no gentamicin was detected in the sera of groups rectally dosed with the test formulations. The results of this study did not provide evidence to support the therapeutic feasibility of rectally absorbed gentamicin.
       
  • Impact of Inadequate Methods and Data Analysis on Reproducibility
    • Abstract: Publication date: February 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 2Author(s): Steve ElliottAbstractFailure to reproduce results of articles is recognized, but the causes, and therefore solutions, are not. One possibility is that deficits in quality of the work result in varying or inconclusive results. Erythropoiesis-stimulating agents have been used to treat anemia in patients with cancer, but there are concerns that erythropoiesis-stimulating agents might stimulate Epo receptors on tumor cells (Epo receptor-cancer hypothesis). Articles have been published on the topic, but the data and conclusions conflict, making them suitable for examination of a relationship between quality and reproducibility. Comprehensive literature searches were performed, and 280 relevant articles were identified. Numerous conflicts between and within these articles were apparent. The incidence of faults in quality parameters was high, including absence of adequate controls (90% of articles), inadequate validation of reagents and methods (87% of articles), and inadequate or improper statistical methods (84% of articles) with questionable interpretation of the data (81% of articles). This resulted in false-positive/negative data that varied with the reagents and methods used. The low quality of evidence may explain the poor reproducibility of Epo receptor-cancer articles.
       
  • Evaluation of Fraction Unbound Across 7 Tissues of 5 Species
    • Abstract: Publication date: February 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 2Author(s): Sangwoo Ryu, David Tess, George Chang, Christopher Keefer, Woodrow Burchett, Gregory S. Steeno, Jonathan J. Novak, Roshan Patel, Karen Atkinson, Keith Riccardi, Li DiAbstractBinding to various tissues and species is frequently assessed in drug discovery and development to support safety and efficacy studies. To reduce time, cost, and labor requirements for binding experiments, we conducted a large comparison study to evaluate the correlation of fraction unbound (fu) across 7 tissues of 5 species, including white adipose, brain, heart, kidney, liver, lung, and skeletal muscle of mouse, rat, dog, monkey, and human. The results showed that there were no significant species differences of fu for tissue binding, and a single-species (e.g., rat) tissue fu can be used as a surrogate for binding in other species. Cross-tissue comparison indicated that brain, heart, liver, and muscle had quite similar fu values; rat liver binding can be used as a surrogate for binding of the other 3 tissues without any scaling factors. Binding to adipose, kidney, and lung can also be estimated with rat liver fu with scaling factors. This study suggests that a single tissue of a single species (e.g., rat liver) is a good predictor for fu of other tissues of various species with or without scaling factors. Molecular size, lipophilicity, pKa, and topological polar surface area are important physiochemical properties influencing tissue fu.
       
  • Pharmacokinetic/Pharmacodynamic Modeling of the Anti-Cancer Effect of
           Dexamethasone in Pancreatic Cancer Xenografts and Anticipation of Human
           Efficacious Doses
    • Abstract: Publication date: February 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 2Author(s): Ye Yao, Qingyu Yao, Yu Fu, Xiuyun Tian, Qiming An, Liang Yang, Hong Su, Wei Lu, Chunyi Hao, Tianyan ZhouAbstractDexamethasone (DEX), a synthetic glucocorticoid, exhibited anti-cancer efficacy in pancreatic xenografts derived from patient tumor tissue or cancer cell lines. The aim of this study was to establish pharmacokinetic/pharmacodynamic (PK/PD) models to quantitatively characterize the inhibitory effect of DEX on tumor growth as well as its discrepancy among 3 xenograft models. Data of tumor growth profiles were collected from a patient-derived xenograft (PDX) model in NOD/SCID mice and 2 cell line–derived (PANC-1 and SW1990) xenograft models in BALB/c nude mice. Empirical PK/PD models were developed to establish mathematical relationships between plasma concentration of DEX and tumor growth dynamics after integrating PK parameters extracted from literature. Drug effect in each model was well described by a linear inhibitory function with a potency factor of 4.67, 3.14, and 2.35 L/mg for PDX, PANC-1, and SW1990 xenograft, respectively. Human efficacious doses of DEX were preliminarily predicted through model-based simulation, and 60% tumor growth inhibition at clinical exposure corresponded to a daily dose range of 26-52 mg intravenously. This modeling work quantified the preclinical anti-cancer effect of DEX and demonstrated the feasibility of its medication in pancreatic cancer, which would be conductive to future translational research.
       
  • Targeted Proteomics-Based Quantitative Protein Atlas of Pannexin and
           Connexin Subtypes in Mouse and Human Tissues and Cancer Cell Lines
    • Abstract: Publication date: February 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 2Author(s): Masanori Tachikawa, Yosuke Kaneko, Sumio Ohtsuki, Yasuo Uchida, Michitoshi Watanabe, Hideo Ohtsuka, Tetsuya TerasakiAbstractThe pannexin (Px) and connexin (Cx) families form multimeric hemichannels that mediate cellular transport of a wide variety of signaling and other molecules and exhibit pathophysiological and pharmacological functions. Twenty-four Px and Cx subtypes have been identified in humans and 23 in mice. The purpose of this study is to establish a quantitative protein atlas of Px and Cx subtypes in mouse and human tissues and cancer cell lines by means of quantitative targeted absolute proteomics, using an internal standard protein in which stable-isotope-labeled target peptides selected according to in silico criteria are concatenated together with internal reference peptides for the determination of the protein amount. This quantification system enabled us to cover 20 of 24 subtypes (83%) in humans, and 21 of 23 subtypes (91%) in mice. In mice, Px1, Cx32, and Cx43 were most abundantly expressed in the small intestine, liver and pancreas, and brain capillary, brain, and heart, respectively. Human blood-brain barrier endothelial cells (human cerebral microvessel endothelial cells) highly expressed Px1 and Cx43. Among human cancer cells, Panc-1 selectively expressed Px1, and Caco-2 cells abundantly expressed Cx32, while MCF-7 and AsPC-1 did not express any subtypes of hemichannels tested. These results suggest that Px1, Cx32, and Cx43 appear to play predominant roles in normal tissues and some cancer cells.
       
  • Enhanced Antipsoriatic Activity of Mycophenolic Acid Against the
           TNF-α-Induced HaCaT Cell Proliferation by Conjugated Poloxamer Micelles
    • Abstract: Publication date: February 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 2Author(s): Wiwat Supasena, Chawanphat Muangnoi, Wuttinont Thaweesest, Chalermkiat Songkram, Keisuke Ueda, Kenjirou Higashi, Kunikazu Moribe, Somboon Tanasupawat, Pornchai RojsitthisakAbstractMycophenolic acid (MPA), an immunosuppressant drug, possesses antimicrobial, anticancer, and antipsoriatic activities. However, the use of MPA in therapeutic applications is limited to its poor oral bioavailability, low aqueous solubility, and undesired gastrointestinal side effects. Polymeric micelles are a drug delivery system that has been used to enhance the water solubility of pharmaceuticals. In this work, poloxamer 407 (P407) and MPA were conjugated via an ester linkage resulting in a P407-MPA conjugate. The P407-MPA conjugate was investigated for micellization, particle size, size distribution, MPA release in phosphate buffer (pH 7.4) and human plasma, and antipsoriatic activity. 1H-nuclear magnetic resonance suggested that polymeric micelles formed from the P407-MPA conjugate exposed its polyethylene oxide chain to the aqueous environment while restricting the conjugated MPA within the inner core. The P407-MPA conjugate has an improved micellization property with the over 12-fold lower critical micelle concentration compared to P407. The conjugate exhibited an enzyme-dependent sustained-release property in human plasma. Finally, the conjugate exhibited an improved antiproliferation activity in tumor necrosis factor-α-induced HaCaT cells, which is an in vitro psoriasis model. Therefore, the prepared P407-MPA conjugate, with an improved aqueous solubility and biological activity of MPA, has the potential to be further developed for psoriasis treatment.
       
  • Impact of Media in Transport Study on Cell Monolayer Integrity and
           Permeability
    • Abstract: Publication date: February 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 2Author(s): Kanyaphat Bunchongprasert, Jun ShaoAbstractLiterature review shows that various media have been used for in vitro transport study, but their impact on the monolayer integrity and permeability has been somehow neglected, and there is no systemic study on this subject. This study aims to investigate the impact of 6 commonly used media on Madin-Darby Canine Kidney cell viability and adherence, and the permeation of mannitol and propranolol. Dulbecco's modified Eagle medium (DMEM), cMedium (DMEM with serum), Hanks’ balanced salt solution (HBSS), HBSS− (without Ca2+/Mg2+), Dulbecco's phosphate-buffered saline (DPBS), and DPBS− (without Ca2+/Mg2+) exhibited no cytotoxicity. Cell detachment was observed in 4 media in the order of DPBS− ≈ HBSS−> DPBS> HBSS. HBSS−/DPBS− caused 70% TEER reduction and 4-fold higher apparent permeability (Papp) of mannitol than the other media. The addition of D-glucose in DPBS− decreased the Papp of mannitol. Cations and glucose negligibly influenced the Papp of propranolol. The addition of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) in HBSS increased the Papp of both mannitol and propranolol for 2-3 folds without TEER reduction. In conclusion, the media compositions (Ca2+/Mg2+, glucose, vitamins, amino acids, and HEPES) can affect the monolayer integrity and permeability. DMEM and cMedium without HEPES are hence suggested as the media in transport study to avoid the potential impact on the monolayer integrity and permeability by the media.
       
  • Chitosan/Sulfobutylether-β-Cyclodextrin Nanoparticles for Ibrutinib
           Delivery: A Potential Nanoformulation of Novel Kinase Inhibitor
    • Abstract: Publication date: February 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 2Author(s): Ludan Zhao, Bin Tang, Peixiao Tang, Qiaomei Sun, Zili Suo, Man Zhang, Na Gan, Huaqing Yang, Hui LiAbstractIn this study, a novel Bruton’s tyrosine kinase inhibitor, ibrutinib, was loaded into chitosan/sulfobutylether-β-cyclodextrin nanoparticles (NPs). NPs have gained high loading efficiency for the hydrophobic drug due to the inclusion of cyclodextrin. Ibrutinib-loaded NPs with an average diameter of 277.9 nm and ζ-potential of +19.1 mV were obtained after regulating several influencing factors. Electrostatic reaction between mucin and NPs indicated that the NPs had a mucoadhesive property. Kinase catalytic phosphorylation was monitored by capillary zone electrophoresis and found that chitosan/sulfobutylether-β-cyclodextrin NPs did not weaken ibrutinib activity on the target kinase. In vitro drug release studies revealed that ibrutinib-loaded NPs exhibited a significantly slower gastric-release rate. This study applied a feasible nanocarrier for ibrutinib delivery, and the potential nanoformulation maintains drug activity and shows a sustained release property. These outcomes are helpful for the formulation exploitation of tyrosine kinase inhibitors.
       
  • Graphene Oxide–Based Nanocomposite for Sustained Release of
           Cephalexin
    • Abstract: Publication date: February 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 2Author(s): Nuwanthi P. Katuwavila, Yasuri Amarasekara, Vimukthi Jayaweera, Chamil Rajaphaksha, Chinthika Gunasekara, Inoka C. Perera, Gehan A.J. Amaratunga, Laksiri WeerasingheAbstractA sustained-release carrier system for the drug cephalexin (CEF) using functionalized graphene oxide is reported. PEGylation of GO (GO-PEG) and successful loading of CEF into PEGylated graphene oxide (GO-PEG-CEF) nanoconjugate are confirmed by Fourier transform infrared spectroscopy, Raman spectroscopy, and thermogravimetric analysis. Encapsulation efficiency of 69% and a loading capacity of 19% are obtained with the optimized formulation of GO-PEG-CEF. In vitro CEF release profiles show an initial burst release followed by a more sustained release over a 96 h period with cumulative release of 80%. The half maximal inhibitory concentration (IC50) values have both dose- and time-dependent antibacterial activity for GO-PEG-CEF against both gram-positive and gram-negative bacteria while pure CEF showed only dose-dependent antibacterial activity. The minimum inhibitory concentration values of GO-PEG-CEF are 7.8 and 3.9 μg/mL against S. aureus and B. cereus, respectively, while it is 10 μg/mL with pure CEF against both gram-positive bacteria. This confirms the enhanced antibacterial activity of GO-PEG-CEF over pure CEF against gram-positive bacteria. These findings therefore show GO-PEG-CEF is promising as a sustained-release nanoantibiotic system for effective treatment against S. aureus and B. cereus infections.
       
  • Suck-Back Impact on Fluid Behavior at Filling Needle Tip
    • Abstract: Publication date: February 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 2Author(s): Maria Clara Novaes Silva, Jean-René Authelin, David Quéré, Charlotte PelletAbstractNeedle clogging induces several issues during the filling step of injectable drugs, which makes essential to avoiding it to ensure a favorable outcome for the process. The suck-back function, present in peristaltic pumps, is often used empirically to that end. This study aims at describing and understanding the fluid behavior after suck-back application, which provides some quantitative specifications to prevent needle clogging.
       
  • Effect of Tensile Stresses on the Evolution of Post-Compaction Properties
           of Sodium Chloride Tablets and Its Mixtures
    • Abstract: Publication date: February 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 2Author(s): Jovana Radojevic, Henrietta Tsosie, Antonios ZavaliangosAbstractThis study focuses on the effect of moisture on the strength of tablets of sodium chloride (NaCl) and its mixtures after compaction. We built on our prior work that proposed an explanation of the strengthening of NaCl tablets due to a dissolution-reprecipitation mechanism and the decrease in strength of NaCl-starch tablets due to the presence of residual stresses on NaCl-NaCl contacts in the mixture. Here, we offer experimental evidence that induced tensile stresses on NaCl slow down or negate (if large enough) the strengthening mechanism. Based on the idea of the negative role of tensile residual stresses in NaCl mixtures, we prove experimentally that NaCl-X mixtures can be optimized in terms of post-compaction strength, if the elastic properties of the second component in the mixture match that of NaCl.
       
  • Role of Surface Characteristics of Mannitol in Crystallization of
           Fenofibrate During Spray Drying
    • Abstract: Publication date: February 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 2Author(s): Poonam Singh Thakur, Samarth D. Thakore, Arvind K. BansalAbstractNanoCrySP™ is a novel spray-drying-based technology for the generation of nanocrystalline solid dispersions of active pharmaceutical ingredients embedded in the matrix of small molecule excipients. Active pharmaceutical ingredient first appears as an amorphous phase, which transforms to crystalline phase during its passage in the drying chamber. Mannitol acts as a crystallization inducer for the intermediate amorphous phase by primary heterogeneous nucleation. Heteronucleation is a surface-assisted phenomenon and surface characteristics of mannitol were hypothesized to play important role. This study investigates the role of surface characteristics of mannitol on crystallization kinetics of amorphous fenofibrate. Crystallization kinetics of amorphous fenofibrate was assessed on 2 surfaces of mannitol having different porosity, roughness, and polarity. Fenofibrate showed faster crystallization in the presence of rougher surface (tind < 1 min) compared with smooth surface (tind = 49.28 min). This was attributed to higher porosity (75%) and surface polarity (~1.25-fold) of rough surface as compared with smooth surface. Polar nature provided primitive sites for faster crystallization of amorphous fenofibrate. These findings can be utilized for generating crystalline solid dispersions using spray drying in the presence of mannitol. The crystalline solid dispersions can be used for the development of oral solid dosage forms.
       
  • Mechanistic Insights of the Critical Role of Hydrogen Donor in Controlling
           Drug Release From Acrylate Adhesive
    • Abstract: Publication date: February 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 2Author(s): Zheng Luo, Chao Liu, Peng Zhang, Peng Quan, Xingzhong Cao, Liang FangAbstractIn the present study, a pyrrolidone adhesive and an amide adhesive were synthesized, and their molecular mechanisms of controlled drug release were described. Using zolmitriptan as model drug, in vitro drug release and skin permeation experiments were performed. Adhesive properties were evaluated using modulated differential scanning calorimetry and rheology study. Free volume of polymer was directly obtained by positron annihilation lifetime spectroscopy. Intermolecular interactions between drugs and adhesives were determined by FTIR spectroscopic analysis and molecular simulation. Release percent (24 h) of zolmitriptan from pyrrolidone adhesive was about 55.8 ± 3.1% (w/w), while from amide adhesive, the release percent (24 h) was about 40.1 ± 1.6% (w/w). The free volume sizes of pyrrolidone adhesive and amide adhesive were about 2309.6 Å3 and 2854.5 Å3, respectively, which were much larger than molecular volume of zolmitriptan (about 285.7 Å3). Thus, the polymer networks might not hinder drug diffusion from the view of free volume. Comparing chemical structures of pyrrolidone group and primary amide group, the main difference was that primary amide group of amide adhesive possessed 2 hydrogen donors. It was proved that hydrogen bonding between zolmitriptan and hydrogen donor of primary amide group played a critical role in controlling drug release.
       
  • Insight From Molecular Dynamics Simulations on the Crystallization
           Tendency of Indomethacin Polymorphs in the Undercooled Liquid State
    • Abstract: Publication date: February 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 2Author(s): Joseph Gerges, Frederic AffouardAbstractThe crystallization tendency of 2 crystalline polymorphs of indomethacin (Iα, Iγ) in the undercooled melt has been investigated using molecular dynamics simulations. The main thermodynamical and dynamical physical parameters involved in the nucleation and growth processes have been determined. A careful attention has been given to the crystal-liquid interfacial free energy which remains really challenging to determine from experiments. The present work particularly sheds the light on the importance of the interplay between the solid-liquid interfacial free energy and the driving force for crystallization. The nucleation and the growth rates have been also estimated in the framework of the classical nucleation theory and some growth modes (normal mode, two-dimensional nucleation, and screw dislocation).
       
  • Polymeric Nanocarriers With Mucus-Diffusive and Mucus-Adhesive Properties
           to Control Pharmacokinetic Behavior of Orally Dosed Cyclosporine A
    • Abstract: Publication date: February 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 2Author(s): Hideyuki Sato, Yuuki Kaneko, Kohei Yamada, Kurt D. Ristroph, Hoang D. Lu, Yoshiki Seto, Hak-Kim Chan, Robert K. Prud’homme, Satomi OnoueAbstractThe present study develops cyclosporine A (CsA)-loaded polymeric nanocarriers with mucus-diffusive and mucus-adhesive potential to control pharmacokinetic behavior after oral administration for the treatment of inflammatory bowel diseases (IBD). CsA-loaded nanocarriers consisting of polystyrene-block-polyethylene glycol (PEG-CsA) and polystyrene-block-polyacrylic acid (PAA-CsA) were prepared by a flash nanoprecipitation. Both nanocarriers showed redispersibility from lyophilized powder back to uniform nanocarrier with a mean diameter of approximately 150 nm. The nanocarriers exhibited significantly improved release behavior of CsA under pH 6.8 condition compared. A test of mucodiffusion, using artificial mucus, demonstrated the mucus-diffusive and mucus-adhesive potential of PEG-CsA and PAA-CsA, respectively, dependent on the lack of electrostatic interactions between the surface-coated polymer and mucin. Oral administrations of PEG-CsA and PAA-CsA (10 mg-CsA/kg) in rats resulted in significant improvements of absorption, as evidenced by 50- and 25-fold higher bioavailability than crude CsA, respectively. PAA-CsA exhibited more sustained and slower absorption process of CsA than PEG-CsA because of the different diffusion behavior within the mucus layer. In the rat model of IBD, significant suppression of inflammatory symptoms could be achieved by oral treatment with both CsA nanoparticles. These polymeric nanocarriers are promising dosage options to control pharmacokinetic behavior of orally dosed CsA, contributing to the development of safe and effective treatment for IBD.
       
  • Surface Tracking of Curcumin Amorphous Solid Dispersions Formulated by
           Binary Polymers
    • Abstract: Publication date: February 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 2Author(s): Na Fan, Tong Lu, Jing LiAbstractHerein, curcumin amorphous solid dispersions (Cur ASDs) were prepared using binary polymers (Eudragit EPO with polyvinylpyrrolidone K30 [EuD-PVP], Eudragit EPO with hydroxypropyl methylcellulose E50 [EuD-HPMC]) as excipient, and surface tracking of Cur ASDs was mainly addressed. Infrared spectroscopy, in situ Raman imaging spectroscopy, molecular docking modeling, and contact angle measurements were mainly applied to study the molecular interaction and wetting property of Cur ASDs. Cur/EuD-PVP had a lower cumulative release (approximately 15%) than Cur/EuD-HPMC (approximately 48%) because Cur/EuD-HPMC presented carrier-controlled wetting property while Cur/EuD-PVP fitted to a drug-controlled wetting property. The favorable HPMC can be preferred as superior excipient for Cur ASDs compared with binary polymers, which has potential application in food and drug healthcare industry of Cur.
       
  • Tunable Two-Compartment On-Demand Sustained Drug Release Based on Lipid
           Gels
    • Abstract: Publication date: February 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 2Author(s): Xiuxia Wang, Liping Huang, Yiwei Zhang, Fanling Meng, Maria Donoso, Roy Haskell, Liang LuoAbstractThe binary-lipid system of soybean phosphatidylcholine (SPC) and glycerol dioleate (GDO) can hydrate to gels on contacting with aqueous mediums, which has emerged as a versatile and promising delivery matrix for extended drug release applications. In the present work, we have characterized the gelation process of this SPC/GDO lyotropic gel (SGLG) system by rheology and evaluated the drug release profiles from the SGLG formulations with different SPC/GDO mass ratios. Our study has demonstrated that simply adjusting the SPC/GDO mass ratio can tune the lipid gelation behavior and modulate the drug release profiles. More importantly, the drug release from the SGLG formulations follows a two-compartment (fast and slow release compartments) release kinetics that has not been reported before. We posit that the fast release compartment corresponds to the passive diffusion of the drug during the early stage of the gel formation. After the boundary gel phase generation, the drug release is then dominated by the slow diffusion process from SGLG. The pharmacokinetic studies in rats match well with the in vitro studies, suggesting that the binary-lipid formulation is an excellent candidate for on-demand sustained drug delivery system.
       
  • Effect of Moisture Sorption on Free Volume and Relaxation of Spray Dried
           Dispersions: Relation to Drug Recrystallization
    • Abstract: Publication date: February 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 2Author(s): Jinjiang Li, Mario Hubert, Swathi Pinnamaneni, Li Tao, Junshu Zhao, Shasad Sharif, Ramesh Krishnan Ramakrishnan, Sergei NazarenkoAbstractThe effect of vapor sorption on the free volume of drug-polymer spray-dried dispersions (SDDs) was investigated, along with the crystallization propensity of drug molecules in SDDs after exposure to humidity. Subsequently, the correlation of free volume change and relaxation time with drug recrystallization was examined. Four polymers, including polyvinylpyrrolidone, polyvinylpyrrolidone vinyl acetate copolymer, hydroxypropyl cellulose, and hydroxypropyl methylcellulose acetate succinate, and 2 drugs (indomethacin and ketoconazole) were selected for preparing SDDs. Free volume data of the exposed SDDs were obtained with positron annihilation lifetime spectroscopy, while the relaxation time was measured using a TA rheometer. Additionally, the crystallization propensity of active pharmaceutical ingredients (APIs) in the exposed SDDs was assessed using both polarized light microscopy and powder X-ray diffraction, followed by relating API crystallization inclination with expansion of holes and relaxation time. Finally, Cohen and Turnbull molecular transport model, along with its extensions by Vrentas and Duda, was qualitatively utilized for interpreting the recrystallization propensity of API molecules. In conclusion, API recrystallization is closely related to free volume change upon moisture sorption and relaxation time, but system dependent; overall, drug-hydroxypropyl methylcellulose acetate succinate SDDs appear physically stable against recrystallization due to less increase in free volume.
       
  • Sensitivity Study to Assess the Robustness of Primary Drying Process in
           Pharmaceutical Lyophilization
    • Abstract: Publication date: February 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 2Author(s): Nirajan Adhikari, Tong Zhu, Feroz Jameel, Ted Tharp, Sherwin Shang, Alina AlexeenkoAbstractThe objective of this work is to apply a sensitivity study to assess the robustness of the primary drying step of pharmaceutical lyophilization with respect to deviations in process parameters. The sensitivity study can provide valuable information regarding the effect of process input parameters on the product quality that can aid in designing robust lyophilization processes. In this study, the output response is related to its inputs using Smolyak sparse grid generalized polynomial chaos method, and the sensitivity was calculated using elementary effects method. Sensitivity of chamber pressure and shelf temperature on product temperature of 2 sucrose-based and one mannitol-based formulation was studied, and the results were analyzed in terms of risk of adverse effects due to process deviations on the product quality. The study revealed that the sensitivity varies among formulations, and preliminary information regarding the possible impact of process deviations can be obtained from the process cycle diagram. The product temperature showed greater sensitivity toward the change in the shelf temperature than toward change in the chamber pressure for the greater part of the primary drying stage. An aggressive process-deviation scenario at the late stage of primary drying was also studied for different formulations, and the results were consistent with the sensitivity study.
       
  • Risk-Based Approach to Lot Release
    • Abstract: Publication date: February 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 2Author(s): Linas Mockus, Gintaras Reklaitis, Kenneth Morris, David LeBlondAbstractIn this work, a novel risk-based methodology for lot release is proposed. Its objective is to assess the risk that a lot declared to have passed truly meets product specifications. The methodology consists of 3 parts: adaptive sample size determination, estimation of the probability that the product was within specifications, and the lot-release decision. The methodology provides a probabilistic statement about the true quality of the batch. Having a probability estimate is the essential condition of risk-based decision-making. We demonstrate the proposed methodology on experimental data generated from 17 immediate-release solid oral drug products from a number of different manufacturers with 5 to 10 lots per manufacturer.
       
  • Investigation of a Microemulsion Containing Clotrimazole and Itraconazole
           for Transdermal Delivery for the Treatment of Sporotrichosis
    • Abstract: Publication date: February 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 2Author(s): Patricia G. Ferreira, Letícia Noronha, Rafaela Teixeira, Italo Vieira, Luana P. Borba-Santos, Alessandra Viçosa, Marcela de Moraes, Sabrina Calil-Elias, Zaida de Freitas, Fernando C. da Silva, Sônia Rozental, Débora O. Futuro, Vitor F. FerreiraAbstractThe aim of this study was to develop a microemulsion (ME) formulation containing an association of itraconazole (ITC) and clotrimazole (CLT) as a transdermal delivery system for the treatment of sporotrichosis. Pseudoternary phase diagrams were constructed to optimize the ME formulation. The ME formulation selected contained 1% (w/w) ITC and 1% (w/w) CLT and was composed of 23.07% Tween® 60 (surfactant), 23.07% propylene glycol (cosurfactant/cosolvent), 30.77% benzyl alcohol (oil), and 21.09% water. The ITC/CLT-loaded ME (ITC/CLT-ME) had a droplet size value of 217 ± 0.9 nm, with a polydispersity index of 0.5 ± 0.1. Permeation experiments on pig ear skin were conducted for ITC/CLT-ME, and the results indicated that the drug permeation performance was influenced by CLT, indicating that CLT acts as a promoter enhancer. In the in vitro antifungal activity assay using Sporothrix brasiliensis yeast, the inhibition halo produced by ITC/CLT-ME exhibited a mean diameter of 43.67 ± 2.31 mm. The ITC/CLT-ME formulation did not cause skin irritation in mice. The results suggest that ITC/CLT-ME is a promising tool for the transdermal treatment of sporotrichosis.
       
  • Effects of Diurnal Variation and Food on Gastrointestinal Transit of
           111In-Labeled Hydrogel Matrix Extended-Release Tablets and 99mTc-Labeled
           Pellets in Humans
    • Abstract: Publication date: February 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 2Author(s): Atsushi Kambayashi, Kazuhiro Sako, Hiromu KondoAbstractThe aim of this research was to characterize the effect of food ingestion and circadian rhythm on the gastrointestinal transit of 2 dosage forms: a hydrogel matrix extended-release (ER) tablet and pellets with the diameter of 9 mm and 150-200 μm, respectively, in humans. Radiolabeled (111In) hydrogel matrix ER tablet and capsule containing radiolabeled (99mTc) pellets were administered with 240 mL of water under the following dosing conditions: fasted state at 8 AM or 8 PM and fed state at 8 AM or 8 PM. A high-fat and high-calorie meal was ingested in the fed state studies. The gastric emptying times and small intestinal transit times of the 2 dosage forms were monitored using gamma scintigraphy. Meal ingestion prolonged gastric emptying times for both dosage forms but had no significant impact on the small intestinal transit times of them. Administration time tended to affect gastric emptying of the ER tablet but had no significant impact on gastric emptying and small intestinal transit for both dosage forms.
       
  • Impact of Hot-Melt Extrusion Processing Conditions on Physicochemical
           Properties of Amorphous Solid Dispersions Containing Thermally Labile
           Acrylic Copolymer
    • Abstract: Publication date: February 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 2Author(s): Alex Mathers, Fatima Hassouna, Lenka Malinová, Jan Merna, Květoslav Růžička, Michal FulemAbstractFor successful formulation of amorphous solid dispersions (ASDs) using hot-melt extrusion, it is imperative to understand the effect that heat and shear rate has on the physicochemical properties of the excipient. In this study, we investigated the influence of hot-melt extrusion parameters on solvent-free binary ASDs of ibuprofen (IBU), a model active pharmaceutical ingredient, in methacrylic acid-ethyl acrylate copolymer type A, 1:1, EUDRAGIT® L100-55 (EUD). To evaluate the impact of barrel temperature, screw speed, and residence time on EUD mass average molar mass and IBU release profile, size-exclusion chromatography and dissolution testing were used, respectively. The optimal conditions were established for IBU loadings less than 40 wt. %. For ASD formulations prepared using the ideal variables, spectral and thermal analyses confirmed that, under dry conditions at a temperature of 25°C, IBU remained amorphous during an 18-month storage period. After 28 months, formulations with active pharmaceutical ingredient content above 30 wt. % started to recrystallize. A temperature–composition phase diagram, constructed using melting point depression and glass-transition temperature measurements of IBU–EUD mixtures, correlated well with the long-term physical stability. The effect that minor-to-moderate polymer degradation within the extrudates has on their long-term physical stability and dissolution characteristics is analyzed and discussed.
       
  • Development of a Pertactin-Coated Beads Approach for Screening of
           Functional Monoclonal Antibodies
    • Abstract: Publication date: February 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 2Author(s): Liwei He, Issaka Yougbare, Beata Gajewska, Jin Su, Rachel Leung, Ali AziziAbstractVaccine manufacturers have recently focused on the development of in vitro potency assays to promote 3R's strategy to replace animal testing. To be able to develop an in vitro potency assay, the immunological characteristics of the monoclonal antibodies used in the assay should be well understood as these antibodies likely reflect the biological activity of a vaccine product. The PRN antigen is one of the immunogenic antigens included in many commercialized acellular pertussis vaccines. Development of an in vitro potency assay for PRN is challenging as the biological properties of PRN are not well understood. In addition, binding of Bordetella pertussis to human cells occurs through multiple bacterial molecules, which makes it very challenging to assess if antibodies contribute to prevention of bacterial adhesion. To overcome these challenges, the functionality of several in-house anti-PRN mAbs has been investigated through a novel approach using PRN-coated beads. We were able to consistently quantify the inhibition of PRN-mediated adhesion for each anti-PRN mAb. Application of the protein-coated beads model has not only enabled screening of functional anti-PRN mAbs but can also be expanded for screening of antibodies against other bacterial or viral antigens.
       
  • Conjugation of Amine-Functionalized Polyesters With Dimethylcasein Using
           Microbial Transglutaminase
    • Abstract: Publication date: February 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 2Author(s): Razan Alaneed, Till Hauenschild, Karsten Mäder, Markus Pietzsch, Jörg KresslerAbstractProtein-polymer conjugates have been used as therapeutics because they exhibit frequently higher stability, prolonged in vivo half-life, and lower immunogenicity compared with native proteins. The first part of this report describes the enzymatic synthesis of poly(glycerol adipate) (PGA(M)) by transesterification between glycerol and dimethyl adipate using lipase B from Candida antarctica. PGA(M) is a hydrophilic, biodegradable but water insoluble polyester. By acylation, PGA(M) is modified with 6-(Fmoc-amino)hexanoic acid and with hydrophilic poly(ethylene glycol) side chains (mPEG12) rendering the polymer highly water soluble. This is followed by the removal of protecting groups, fluorenylmethyloxycarbonyl, to generate polyester with primary amine groups, namely PGA(M)-g-NH2-g-mPEG12. 1H NMR spectroscopy, FTIR spectroscopy, and gel permeation chromatography have been used to determine the chemical structure and polydispersity index of PGA(M) before and after modification. In the second part, we discuss the microbial transglutaminase–mediated conjugation of the model protein dimethylcasein with PGA(M)-g-NH2-g-mPEG12 under mild reaction conditions. SDS-PAGE proves the protein-polyester conjugation.
       
  • Evaluating Heparin Products for Heparin-Induced Thrombocytopenia Using
           Surface Plasmon Resonance
    • Abstract: Publication date: February 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 2Author(s): Fuming Zhang, Payel Datta, Jonathan S. Dordick, Robert J. LinhardtAbstractHeparin-induced thrombocytopenia (HIT) is an adverse immunological disorder caused by antibodies to platelet factor 4 (PF4)-heparin complexes. The analysis of HIT potential for different heparin and heparin-related products is important prior to their clinical application. Here, we report a rapid method for the evaluation of HIT potential of various heparin and heparin-related compounds using surface plasmon resonance (SPR). Solution competition between surface-immobilized heparin and soluble unfractionated heparin, low molecular weight heparin (LMWH), or ultra-LMWH binding to PF4 was performed using SPR to measure the half maximal inhibitory concentration (IC50) of different heparin products. The IC50 values of different unfractionated heparin active pharmaceutical ingredients (APIs) varied from 0.38 to 0.6 μg/mL and the IC50 values of different LMWH APIs ranged from 2.4 to 2.9 μg/mL. The IC50 of Arixtra® (a synthetic pentasaccharide ultra-LMWH) was not measurable even at very high concentrations. These differences in IC50 values for different heparin products suggest a quantitative means for evaluating the HIT potential of various heparins and heparin-related products.
       
  • Polysialic Acid-Mediated Activity Measurement of Polysialylated
           Recombinant Coagulation Factor VIII
    • Abstract: Publication date: February 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 2Author(s): Alfred Weber, Andrea Engelmaier, Jürgen Siekmann, Peter L. TurecekAbstractMeasurement of modified biologic including coagulation factors with extended half-life obtained, for example, by polysialylation pose an analytical challenge especially if both biological activity and presence of modification have to be determined. Analytical methods applied so far address only 1 of the 2 quality attributes of modified biologics. Here, we describe the development and bioanalytical validation of a polysialic acid-mediated factor VIII activity assay: Polysialic acid-specific capture of polysialylated recombinant factor VIII is combined with a chromogenic FVIII activity test using commercially available reagents. This assay principle enabled measurement of FVIII activity down to the pico mole-range without any interference by nonmodified factor VIII. To the best of our knowledge, this is the first method to selectively, accurately, and precisely measure simultaneously activity and modification integrity of a polysialylated biologic in complex matrices, as shown by the bioanalytical validation data. The convenience, robustness, and reliability of using this method has been demonstrated by its application for the nonclinical development of the polysialylated recombinant FVIII preparation. The method principle could be applied to protein modifications other than polysialylation and to activity tests other than the chromogenic FVIII assay.
       
  • Production Process Development of Pseudorabies Virus Vaccine by Using a
           Novel Scale-Down Model of a Fixed-Bed Bioreactor
    • Abstract: Publication date: February 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 2Author(s): Jianqi Nie, Yang Sun, Feng Peng, Xinran Li, Yankun Yang, Xiuxia Liu, Ye Li, Chunli Liu, Zhonghu BaiAbstractIn this study, a novel tube-fixed-bed bioreactor which consists of a TubeSpin bioreactor 50 tube and 0.44 g macrocarriers was developed as the scale-down model of a fixed-bed bioreactor. The adherent Vero cell–based pseudorabies virus (PRV) production process was tested in this novel model. The Vero cells grew well in the tube-fixed-bed bioreactor, and the cell density reached 5.8 × 106 cells/mL after 7 days of culture. The PRV production parameters (time of infection, multiplicity of infection, and harvest process) were optimized in the tube-fixed-bed bioreactor. Then the optimized process (time of infection = 3 days, multiplicity of infection = 0.001 and multiple harvest process) was scaled up 25-fold to an Xcell 1-L laboratory-scale fixed-bed bioreactor and 125-fold to an Xcell 5-L fixed-bed bioreactor successfully. The total PRV harvest in the Xcell 1-L bioreactor at 5 days after infection (dpi) was 10.25 log10 TCID50 which corresponds to 177,827 doses of vaccine. The total PRV harvest in the Xcell 5-L bioreactor at 5 dpi was 11.13 log10 TCID50 which corresponded to 1,348,962 doses of vaccine. The comparable growth curve, metabolism, and PRV production profile of the scaled-up bioreactors confirmed the feasibility and scalability of the tube-fixed-bed bioreactor as a scale-down model of the fixed-bed bioreactor for virus production process development.
       
  • Preformulation Studies and Enabling Formulation Selection for an Insoluble
           Compound at Preclinical Stage—From In Vitro, In Silico to In Vivo
    • Abstract: Publication date: February 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 2Author(s): Lijun Zhang, Hansen Luan, Weiyue Lu, Hao WangAbstractThe objective of this work was to identify an enabling formulation for an insoluble compound ZL006 with potency of boosting leukocytes after chemotherapy. The low oral bioavailability (
       
  • Commentary on the Benefits of US Pharmacopeial Standards: A Generic
           Pharmaceutical Industry Survey
    • Abstract: Publication date: February 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 2Author(s): Ian K. Warthin, Julia Berik, Doug Podolsky, Vimala Raghavendran, Ravi Reddy, Judy Chang, Noah Porter, Nicholas GaritoAbstractPharmacopeial quality standards for medicines are associated with ensuring quality, but they can also facilitate innovation and competition in pharmaceutical markets. Unpublished data suggest that pharmacopeial quality standards, while not associated with increasing the likelihood of a first generic entrant in the market, are associated with increasing the overall number of generic entrants. This commentary describes an online survey of finished-dose generic drug manufacturers in the United States and India that explores whether using pharmacopeial documentary standards and physical reference standards (RSs) such as those developed by the US Pharmacopeial Convention, accelerate generic drug development and reduce the duration, cost, and risk involved in bringing generic drugs to the US market. The survey provides new evidence that using pharmacopeial documentary standards and physical RSs (1) saves generic drug companies 19% of the time spent on generic product development overall and 31% of time spent on analytical method development and (2) provides clear guidance on quality, thereby reducing the perceived risk of Abbreviated New Drug Application rejection by the US Food and Drug Administration. Ninety percent of respondents agreed that the use of pharmacopeial documentary standards accelerated product development compared with 85% for physical RSs; and 92% of respondents agreed that the use of documentary standards and physical RSs reduced the risk that their Abbreviated New Drug Application would be rejected. Owing to the relatively small sample size of the survey, the results should be viewed as entirely directional for use in trends analysis on the finished-dose generic pharmaceutical industry. The margin of error for all results is 8%-14% at a 95% confidence interval.
       
  • A Wish List for Drug Development in Pediatrics
    • Abstract: Publication date: February 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 2Author(s): Rachel MeyersAbstractThis commentary illustrates and summarizes some of the many issues with dosage forms and drug delivery that arise in everyday practice for clinicians caring for pediatric patients. While advances in drug development for children and expansion of labeling information in this age group have made great strides in improving care, there is much to be desired in the area of dosage forms. From liquids that are unpalatable to the need for extemporaneous compounding of parenteral doses in syringes that are meant for immediate use but used to store medications for hours to weeks, the list of challenges is long. Many of these problems exist for drugs which have long been generic, but some problems have arisen from new drugs as well. Pediatric clinicians and dosage form developers should work together to create solutions.
       
  • Editorial
    • Abstract: Publication date: February 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 2Author(s): Ronald T. Borchardt
       
  • Editorial
    • Abstract: Publication date: February 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 2Author(s): Ronald T. Borchardt
       
  • Editorial Advisory Board
    • Abstract: Publication date: February 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 2Author(s):
       
  • Dietary-Induced Obesity, Hepatic Cytochrome P450, and Lidocaine
           Metabolism: Comparative Effects of High-Fat Diets in Mice and Rats and
           Reversibility of Effects With Normalization of Diet
    • Abstract: Publication date: February 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 2Author(s): Hamdah M. Al Nebaihi, Rami Al Batran, John R. Ussher, Zaid H. Maayah, Ayman O.S. El-Kadi, Dion R. BrocksAbstractThe effects of a high-fat diet on mRNA and protein of cytochrome P450 (CYP) enzymes in rats and mice and its impact on lidocaine deethylation to its main active metabolite, monoethylglycinexylidide (MEGX), in rats were investigated. The effect of a change in diet from high-fat to standard diet was also evaluated. Plasma biochemistry, mRNA, protein expression for selected CYP, and the activity of lidocaine deethylation were determined. The high-fat diet curtailed the activity and the expression of the majority of CYPs (CYP1A2, CYP3A1, CYP2C11, CYP2C12, and CYP2D1), mRNA levels (Cyp1a2 and Cyp3a2), and MEGX maximal formation rate (Vmax). Mice showed complementary results in their protein expressions of cyp3a and 1a2. Switching the diet back to standard chow in rats for 4 weeks reverted the expression levels of mRNA and protein back to normal levels as well as the maximum formation rates of MEGX. Female and male rodents showed similar patterns in CYP expression and lidocaine metabolism in response to the diets, although MEGX formation was faster in male rats. In conclusion, diet-induced obesity caused general decreases in CYP isoforms not only in rats but also in mice. The effects were shown to be reversible in rats by normalizing the diet.
       
  • Prediction of precision for purity methods
    • Abstract: Publication date: Available online 21 January 2020Source: Journal of Pharmaceutical SciencesAuthor(s): Izydor Apostol, Richard Wu, Mee Ko, Jiu-Li Song, Lan Li, Gregory Schlobohm, Wojciech SzpankowskiAbstractMethods that determine the relative purity of biopharmaceuticals represent the most widely used form of analysis for the pharmaceutical industry. The ability to rapidly assess method capability, or the uncertainty of measurements, under actual use conditions continues to present significant challenges. We have refined and applied the model of Uncertainty Based on Current Information (UBCI) to predict the precision of the purity measurements and compared the predicted precision to the statistically derived variability for several different types of purity methods. The determination of the statistically derived method variability was derived from the analysis of data sets ranging from hundreds to thousands of measurements for each different method type. The predicted precision was found to be in excellent agreement with the statistically obtained values with R2=0.94. This demonstration of concurrence between the predicted and actual precision provides an opportunity for streamlining laborious conventional (statically derived) assessment of method precision and leveraging the UBCI model utilizing much smaller data sets or even a single experiment.
       
  • Engineering Stability, Viscosity, and Immunogenicity of Antibodies by
           Computational Design
    • Abstract: Publication date: Available online 18 January 2020Source: Journal of Pharmaceutical SciencesAuthor(s): Daisuke Kuroda, Kouhei TsumotoAbstractIn recent years, computational methods have garnered much attention in protein engineering. A large number of computational methods have been developed to analyze the sequences and structures of proteins and have been used to predict the various properties. Antibodies are one of the emergent protein therapeutics, and thus methods to control their physicochemical properties are highly desirable. However, despite the tremendous efforts of past decades, computational methods to predict the physicochemical properties of antibodies are still in their infancy. Experimental validations are certainly required for real-world applications, and the results should be interpreted with caution. Among the various properties of antibodies, we focus in this review on stability, viscosity, and immunogenicity, and we present the current status of computational methods to engineer such properties.
       
  • Effects of tubing type, operating parameters and surfactants on particle
           formation during peristaltic filling pump processing of a monoclonal
           antibody formulation
    • Abstract: Publication date: Available online 16 January 2020Source: Journal of Pharmaceutical SciencesAuthor(s): Cheng Her, Laura Tanenbaum, Swati Bandi, Theodore Randolph, Renuka Thirumangalathu, Krishna Mallela, John Carpenter, Yannick EliasAbstractFilling pump operation is an important cause of particle formation in therapeutic protein formulations. The goals of the current study were to investigate the impacts of peristaltic filling pump tubing type, pump operating parameters (acceleration and velocity) and formulation on both nano- and microparticle formation in water, placebo and a 120 mg/ml monoclonal antibody (mAb) drug formulation. Microparticles were quantified using flow imaging microscopy and light obscuration, and nanoparticles were counted with nanoparticle tracking analysis (NTA). Pumping of all solutions through Pharmed® tubing resulted in much higher particle levels than processing with Accusil™ or Masterflex® tubing. Pump acceleration did not measurably affect particle levels in pumped solutions. But in some cases, a relatively high pumping velocity of 400 rpm enhanced nanoparticle formation. The presence of surfactants reduced pumping-induced particle formation in the mAb solution, and the effects of four different surfactants tested were similar. Biophysical properties (secondary and tertiary structure, and thermal stability) of the protein in solution were not altered by pumping. Overall, this study demonstrates that investigations of pumping parameters and formulations using both nano- and microparticle measurement methods are important for understanding pumping-induced particle formation and developing effective control strategies.
       
  • The role of Self Nano Emulsifying drug delivery systems of CDODA-Me in
           sensitizing Erlotinib resistant Nonsmall cell lung cancer
    • Abstract: Publication date: Available online 15 January 2020Source: Journal of Pharmaceutical SciencesAuthor(s): Ebony Nottingham, Vasanthakumar Sekar, Arindam Mondal, Stephen Safe, ArunK. Rishi, Mandip SinghAbstractIn this study we have investigated the effects of combination treatment involving ERL (Erlotinib) with a glycyrrhetinic acid analog, CDODA-Me in overcoming ERL resistance, and to improve the oral bioavailability of this treatment using self-Nano-emulsifying drug delivery systems (SNEDDS). A Qbd (quality by design) approach was used to prepare CDMS (CDODA-SNEDDS, 2μΜ), which were characterized using surface response methodology to optimize drug content, particle size, as well as drug release. CDMS/ERL combinations showed synergism in wild type and resistant H1975 and HCC827 cell lines with combination index values less than 1. Increased apoptosis, mitochondrial membrane potential depletion and enhanced intracellular ROS levels were also observed in combination therapy. Western blot analysis showed that combination therapy inhibited phosphorylation of epidermal growth factor receptor (EGFR) (p
       
  • Carfilzomib delivery by quinic acid-conjugated nanoparticles: Discrepancy
           between tumoral drug accumulation and anticancer efficacy in a murine 4T1
           orthotopic breast cancer model
    • Abstract: Publication date: Available online 13 January 2020Source: Journal of Pharmaceutical SciencesAuthor(s): Yearin Jun, Jun Xu, Hyungjun Kim, Ji Eun Park, Yoo-Seong Jeong, Jee Sun Min, Naeun Yoon, Ji Yoon Choi, Jisu Yoo, Soo Kyung Bae, Suk-Jae Chung, Yoon Yeo, Wooin LeeAbstractDespite being a major breakthrough in multiple myeloma therapy, carfilzomib (CFZ, a second-generation proteasome inhibitor drug) has been largely ineffective against solid cancer, possibly due to its pharmacokinetic drawbacks including metabolic instability. Recently, quinic acid (QA, a low-affinity ligand of selectins upregulated in peritumoral vasculature) was successfully utilized as a surface modifier for nanoparticles containing paclitaxel. Here, we designed QA-conjugated nanoparticles containing CFZ (CFZ@QANP; the surface of poly(lactic-co-glycolic acid) (PLGA) nanoparticles modified by conjugation with a QA derivative). Compared to the clinically used cyclodextrin-based formulation (CFZ-CD), CFZ@QANP enhanced the metabolic stability and in vivo exposure of CFZ in mice. CFZ@QANP however showed little improvement in suppressing tumor growth over CFZ-CD against the murine 4T1 orthotopic breast cancer model. CFZ@QANP yielded no enhancement in proteasomal inhibition in excised tumors despite having a higher level of remaining CFZ than CFZ-CD. These results likely arise from delayed, incomplete CFZ release from CFZ@QANP as observed using biorelevant media in vitro. These results suggest that the applicability of QANP may not be predicted by physicochemical parameters commonly used for formulation design. Our current results highlight the importance of considering drug release kinetics in designing effective CFZ formulations for solid cancer therapy.
       
  • Exploring poly(ethylene glycol)-poly(trimethylene carbonate) nanoparticles
           as carriers of hydrophobic drugs to modulate osteoblastic activity
    • Abstract: Publication date: Available online 11 January 2020Source: Journal of Pharmaceutical SciencesAuthor(s): Diana M. Leite, Daniela M. Sousa, Meriem Lamghari, Ana Paula PêgoAbstractCurrent treatment options for bone-related disorders rely on a systemic administration of therapeutic agents that possess low solubility and/or intracellular bioavailability and a high pharmacokinetic variability, which lead to major off-target side effects. Hence, there is an unmet need of developing drug delivery systems that can improve the clinical efficacy of such therapeutic agents. Nanoparticle delivery systems might serve as promising carriers of hydrophobic molecules. Here, we propose two nanoparticle-based delivery systems based on monomethoxy poly(ethylene glycol)-poly(trimethyl carbonate) (mPEG-PTMC) and poly(lactide-co-glycolide) (PLGA) for the intracellular controlled release of a small hydrophobic drug (dexamethasone, DEX) to osteoblast cells in vitro. mPEG-PTMC self-assembles into stable nanoparticles in the absence of surfactant and shows a greater entrapment capacity of DEX, while assuring bioactivity in MC3T3-E1 and BMSCs cultured under apoptotic and osteogenic conditions, respectively. The mPEG-PTMC nanoparticles represent a potential vector for the intracellular delivery of hydrophobic drugs in the framework of bone-related diseases.
       
  • Kinetics and competing mechanisms of antibody aggregation via bulk and
           surface-mediated pathways
    • Abstract: Publication date: Available online 10 January 2020Source: Journal of Pharmaceutical SciencesAuthor(s): Caitlin V. Wood, Sean McEvoy, Vladimir I. Razinkov, Wei Qi, Eric M. Furst, Christopher J. RobertsAbstractNon-native protein aggregation is a long-standing obstacle in the biopharmaceutical industry. Proteins can aggregate through different mechanisms, depending on the solution and stress conditions. Aggregation in bulk solution has been extensively studied in a mechanistic context and is known to be temperature-dependent. Conversely, aggregation at interfaces has been commonly observed for liquid formulations but is less understood mechanistically. This work evaluates the combined effects of temperature and compression/dilation of air-water interfaces on aggregation rates and particle formation for anti-streptavidin (AS) immunoglobulin gamma-1. Aggregation rates are quantified via size exclusion chromatography, dynamic light scattering, and micro-flow imaging as a function of temperature and extent of air-liquid interface compressions. Competition exists between bulk- and surface-mediated aggregation mechanisms. Each has a largely different temperature dependence that leads to a crossover between the dominant aggregation mechanism as the sample temperature changes. Surface-mediated aggregation rates are pH-dependent and correlate with electrostatic protein-protein interactions, but do not mirror the pH-dependence of bulk aggregation rates that instead follow trends for conformational stability. Mechanistic insights were informed by quiescent incubation of solutions before and after interface compressions. Detailed mechanistic conclusions require direct dynamic observation at the interface. Microbubble tensiometry is introduced as a promising tool for such measurements.
       
  • Impact of Formulation and Suspension Properties on Redispersion of
           Aluminum Adjuvanted Vaccines
    • Abstract: Publication date: Available online 10 January 2020Source: Journal of Pharmaceutical SciencesAuthor(s): Alex Langford, Teresa Horwitz, Emmanuel Adu-Gyamfi, Chris Wiley, Elizabeth Holding, Daniel Zimmermann, Arun Alphonse Ignatius, Satoshi OhtakeAbstractThe adsorption of antigens to the surface of two commonly used insoluble adjuvants, aluminum phosphate and aluminum hydroxide, has been well characterized. In spite of the pharmaceutical benefits, alum-based vaccine formulations can present challenges in redispersion of the final product after storage. Inability to resuspend alum-based vaccines during administration results in inadequate dosing, thus rendering the product unusable. Here, the influence of formulation conditions on the resuspendability of aluminum adjuvant-containing vaccines was investigated. Particle size analysis by Micro-Flow Imaging (MFI™), zeta potential measurement, and sedimentation analysis by Turbiscan® were employed to characterize suspension properties. Ionic strength, pH, and antigen concentration were found to significantly influence sedimentation behavior, particle size, and redispersion. Increasing ionic strength increased the sedimentation rate of adjuvants favoring resuspendability. The addition of BSA to aluminum phosphate reduced resuspendability more significantly than the addition of lysozyme. Decreased resuspendability correlated with an increase in fine-to-large particle ratio and decrease in sedimentation rate. In summary, resuspendability of adjuvant drug product is favored by increased flocculation, decrease in fine-to-large particle ratio, and reduction in surface charge of antigen and adjuvant. A careful balance of these formulation conditions can therefore be an effective means to mitigate challenges of alum adjuvant redispersion.
       
  • Predicting the Solubility Advantage of Amorphous Drugs: Effect of pH
    • Abstract: Publication date: Available online 10 January 2020Source: Journal of Pharmaceutical SciencesAuthor(s): Peter J. Skrdla, Philip D. Floyd, Philip C. Dell’OrcoAbstractThe solubility enhancement generated by an amorphous phase over its crystalline counterpart is unaffected by the pH of solution (at fixed ionic strength and temperature), even for a drug containing ionizable moieties, provided that both the ionized and neutral species generated through dissolution of the solids remain dissolved.
       
  • Diclofenac prodrugs for intra-articular depot injectables: In vitro
           hydrolysis and species variation
    • Abstract: Publication date: Available online 10 January 2020Source: Journal of Pharmaceutical SciencesAuthor(s): Ida Hagen Storgaard, Jesper Kristensen, Claus Larsen, Nina Mertz, Jesper Østergaard, Susan Weng LarsenAbstractIntra-articular depot injectables based on in situ suspension formation of ester prodrugs of non-steroidal anti-inflammatory drugs (NSAIDs) are promising for management of joint pain. As candidates for this delivery approach, five diclofenac ester prodrugs comprising different imidazole-containing promoieties were synthesized and their physicochemical properties characterized. In vitro hydrolysis rates were investigated in buffer solutions, in 40% (v/v) human, equine, canine, and rat plasma, and in 80% (v/v) human and equine synovial fluid. Bioconversion of the prodrugs to diclofenac was found to be enzyme-mediated and follow pseudo-first-order kinetics. Large variations in hydrolysis rates were observed between species and between prodrugs, with prodrug half-lives in plasma from canine, rat, horse and human of 3.44 – 141 min, 2.51 – 14 min, 0.58 – 1.31 min, and 0.23 – 1.70 min, respectively. Half-lives in human and equine synovial fluid were 1.6 to 28-fold larger than in plasma. The results highlight the significance of species as well as tissue variation in prodrug design and suggest that the horse may constitute a suitable model for testing the intra-articular depot approach. Two prodrug candidates appeared promising for future in vivo studies based on their rapid in vitro enzyme-mediated bioconversion to diclofenac and physiochemical characteristics.
       
  • An NMR Based Similarity Metric for Higher Order Structure Quality
           Assessment among U.S. Marketed Insulin Therapeutics
    • Abstract: Publication date: Available online 9 January 2020Source: Journal of Pharmaceutical SciencesAuthor(s): Deyun Wang, Junyong Park, Sharadrao M. Patil, Cameron J. Smith, John L. Leazer, David A. Keire, Kang ChenAbstractProtein or peptide higher order structure (HOS) is a quality attribute that could affect therapeutic efficacy and safety. Where appropriate, the HOS similarity between a proposed follow-on product and the reference listed drug (RLD) should be demonstrated during regulatory assessment. Establishing quantitative HOS similarity for two drug substances (DS), manufactured by different processes, has been challenging. Herein, HOS differences among US marketed insulin drug products (DP) were quantified using NMR spectra and principal component analysis (PCA). Then, the unitless Mahalanobis distance (DM) in PCA space was calculated between insulin analog RLDs and their recently approved follow-on products and all DM values were 3.29 or less. By contrast, a larger DM value of 20.5 was obtained between the two Insulin Human DPs independently approved. However, upon mass-balanced and reversible dialysis of the two Insulin Human DPs against the same buffers, the DM value was reduced to 1.19 or less. Thus, the observed range of NMR-PCA derived DM values can be used as a robust and sensitive measure of HOS similarity. Overall, the DM values of 3.3 for DP and 1.2 for DS using insulin therapeutics represented realistic and achievable similarity metrics for developing generic or biosimilar drugs, quality assurance or control.
       
  • Excipients for Room Temperature Stable Freeze-Dried Monoclonal Antibody
           Formulations
    • Abstract: Publication date: January 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 1Author(s): Christina Haeuser, Pierre Goldbach, Joerg Huwyler, Wolfgang Friess, Andrea AllmendingerAbstractSucrose is a common cryoprotectant and lyoprotectant to stabilize labile biopharmaceuticals during freeze-drying and storage. Sucrose-based formulations require low primary drying temperatures to avoid collapse and monoclonal antibody (mAb) containing products need to be stored refrigerated. The objective of this study is to investigate different excipients enabling storage at room temperature and aggressive, shorter lyophilization cycles. We studied combinations of 2-hydroxypropyl-beta-cyclodextrin (CD), recombinant human albumin, polyvinylpyrroldione (PVP), dextran 40 kDa (Dex), and sucrose (Suc) using 2 mAbs. Samples were characterized for collapse temperature (Tc), glass transition temperature of the liquid (Tg′) and freeze-dried formulation (Tg), cake appearance, residual moisture, and reconstitution time. Freeze-dried formulations were stored at 5°C, 25°C, and 40°C for up to 9 months and mAb stability was analyzed for color, turbidity, visible and sub-visible particles, and monomer content. Formulations with CD/Suc or CD/PVP/Suc were superior to pure Suc formulations for long-term storage at 40°C. When using aggressive freeze-drying cycles, these formulations were characterized by pharmaceutically elegant cakes, short reconstitution times, higher Tg′, Tc, and Tg. We conclude that the addition of CD allows for shorter freeze-drying cycles with improved cake appearance and enables storage at room temperature, which might reduce costs of goods substantially.
       
  • Development of Freeze-Drying Cycles for Pharmaceutical Products Using a
           Micro Freeze-Dryer
    • Abstract: Publication date: January 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 1Author(s): Davide Fissore, Maite Harguindeguy, Daniela Velez Ramirez, Taylor N. ThompsonAbstractThis article aims to investigate how a small-scale freeze-dryer can be used for process design. The system encompasses a temperature controlled metallic ring that surrounds a small batch of vials, in contact with the external vials through removable thermal conductors. The temperature of the ring can be modified to keep a constant difference with the temperature of one or more vials of the batch. In this article, an extensive validation of the system is given, considering 10% w/w sucrose and 5% w/w mannitol solutions, processed in different types of vials (6 R and 20 R) and in different operating conditions. The micro freeze-dryer was also shown to be able to provide accurate estimates of the overall heat transfer coefficient from the shelf to the product in the vials (Kv) and of the resistance of the dried cake to vapor flux (Rp): both values appeared to be very close to those obtained for the same case studies in a pilot-scale unit. Finally, the use of the micro freeze-dryer to control product temperature and drying time values to simulate a pilot-scale unit was addressed, thus demonstrating the adequacy of this system for process scaleup.
       
  • An Experimental-Based Approach to Construct the Process Design Space of a
           Freeze-Drying Process: An Effective Tool to Design an Optimum and Robust
           Freeze-Drying Process for Pharmaceuticals
    • Abstract: Publication date: January 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 1Author(s): Getachew Assegehegn, Edmundo Brito-de la Fuente, José M. Franco, Críspulo GallegosAbstractThe application of quality by design (QbD) is becoming an integral part of the formulation and process development for pharmaceutical products. An essential feature of the QbD philosophy is the design space. In this sense, a new approach to construct a process design space (PDS) for the primary drying section of a freeze-drying process is addressed in this paper. An effective customized design of experiments (DoE) is developed for freeze-drying experiments. The results obtained from the DoE are then used to construct the product-based PDS. The proposed product-based PDS construction approach has several advantages, including (1) eliminating assumptions on the heat transfer coefficient and dried product resistance, as it is constructed from experimental results specifically obtained from a given formulation, yielding more realistic and reliable results and (2) PDS construction based on a narrow range of product temperatures and considering the variations in product temperature and sublimation rate of vials across a shelf. This guarantees the effectiveness and robustness of the process and facilitates the process scale-up and transfer. The PDS developed herein was experimentally verified. The PDS predicted parameters were in excellent agreement with the experimentally obtained parameters.
       
  • The Glucagon-Like Peptide 2 Analog Teduglutide Reversibly Associates to
           Form Pentamers
    • Abstract: Publication date: January 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 1Author(s): John S. Philo, Wasyl Sydor, Tsutomu ArakawaAbstractGlucagon-like peptide 1 and 2 and their analog peptide therapeutics are known to reversibly associate to form oligomers. Here we report the association properties of the glucagon-like peptide 2 analog teduglutide at concentrations up to ∼15 mg/mL. Both sedimentation equilibrium (SE-AUC) and sedimentation velocity (SV-AUC) show that teduglutide dissociates completely to monomers below 0.1 mg/mL. SE-AUC shows that the apparent weight-average molar mass increases substantially between 0.1 and 1 mg/mL, reaching a maximum of ∼14.5 kDa (∼3.9-mer) near 2 mg/mL, and then falling at higher concentrations because of strong solution nonideality effects (highly positive second virial coefficient). Circular dichroism spectra over the range from 0.1 to 2 mg/mL show that self-association is accompanied by significant increases in alpha-helix content, and that the associated state has a distinct tertiary structure. The SV-AUC data up to 2.2 mg/mL are fitted fairly well by an ideal rapidly reversible monomer-pentamer association. The SE-AUC modeling included thermodynamic nonideality effects. SE-AUC data up to ∼15 mg/mL imply a monomer-pentamer association at lower concentrations, but the pentamers also appear to weakly associate to form decamers. These results illustrate the importance of directly modeling the solution nonideality effects, which if neglected would lead to an incorrect preferred stoichiometry.
       
  • Expedited Investigation of Powder Caking Aided by Rapid 3D Prototyping of
           Testing Devices
    • Abstract: Publication date: January 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 1Author(s): Cosima Hirschberg, Johan Boetker, Jens Risbo, Changquan Calvin Sun, Jukka RantanenAbstractPowder caking can dramatically affect powder handling and downstream production processes. Understanding the key factors that contribute to bulk powder caking is crucial. This article introduces the Hirschberg caking device (HCD), which is a 3D-printed device allowing for parallel testing of powder caking in a cylindrical geometry. In the HCD setup, the powder sample is stored in controlled conditions in the sample holder. On removal of the sample holder, the caked powder will remain in the shape determined by the sample geometry while the remaining powder will fall down. Caking indices can be calculated based on image analysis and weight measurement. The results obtained for the caking of lactose monohydrate with the HCD were in good agreement with the results obtained by a ring shear tester. In addition, a strain tester was used to measure the strength of the formed cakes. Using this approach, critical storage conditions and the required concentration of a given anticaking agent (talc) for lactose monohydrate could be identified. This work demonstrates the potential of rapid prototyping in powder characterization by introducing a fast and affordable approach for exploring and trouble-shooting powder caking.
       
  • Overcoming Challenges of Implementing Closed System Transfer Device
           Clinical In-Use Compatibility Testing for Drug Development of Antibody
           Drug Conjugates
    • Abstract: Publication date: January 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 1Author(s): Frankie Petoskey, Stanley C. Kwok, William Jackson, Shan JiangAbstractClosed system transfer devices (CSTD) are a supplemental engineering control designed to reduce occupational exposure of hazardous drugs and are currently implemented in accordance with evolving regulations. Owing to the novelty and complexity of these devices and their importance in clinical in-use testing, here we evaluated FDA-approved CSTD, assessing product quality through stability indicating assays to determine any drug product incompatibilities. Six devices were used in a simulated compounding and administration of a late-phase IgG1 antibody-drug conjugate (ADC) and the resulting samples were analyzed for visible and subvisible particle counts by light obscuration and micro–flow imaging, physical stability by size exclusion chromatography, and biological activities by relative potency. Potential challenges included improper fit of CSTD components, loss of product to void volume, and material incompatibility. Results showed compatibility of the ADC with the 6 CSTD evaluated. One CSTD introduced subvisible particles into the ADC during compounding that were identified through morphological assessment as silicone oil. This study highlights the importance of clinical in use testing with new devices and proposes strategies to mitigate the risk of drug product incompatibility with CSTD.
       
  • Activation of Human Monocytes by Colloidal Aluminum Salts
    • Abstract: Publication date: January 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 1Author(s): Hilde Vrieling, Sietske Kooijman, Justin W. de Ridder, Dominique M.E. Thies-Weesie, Peter C. Soema, Wim Jiskoot, Elly van Riet, Albert J.R. Heck, Albert P. Philipse, Gideon F.A. Kersten, Hugo D. Meiring, Jeroen L. Pennings, Bernard MetzAbstractSubunit vaccines often contain colloidal aluminum salt–based adjuvants to activate the innate immune system. These aluminum salts consist of micrometer-sized aggregates. It is well-known that particle size affects the adjuvant effect of particulate adjuvants. In this study, the activation of human monocytes by hexagonal-shaped gibbsite (ø = 210 ± 40 nm) and rod-shaped boehmite (ø = 83 ± 827 nm) was compared with classical aluminum oxyhydroxide adjuvant (alum). To this end, human primary monocytes were cultured in the presence of alum, gibbsite, or boehmite. The transcriptome and proteome of the monocytes were investigated by using quantitative polymerase chain reaction and mass spectrometry. Human monocytic THP-1 cells were used to investigate the effect of the particles on cellular maturation, differentiation, activation, and cytokine secretion, as measured by flow cytometry and enzyme-linked immunosorbent assay. Each particle type resulted in a specific gene expression profile. IL-1ß and IL-6 secretion was significantly upregulated by boehmite and alum. Of the 7 surface markers investigated, only CD80 was significantly upregulated by alum and none by gibbsite or boehmite. Gibbsite hardly activated the monocytes. Boehmite activated human primary monocytes equally to alum, but induced a much milder stress-related response. Therefore, boehmite was identified as a promising adjuvant candidate.
       
  • Effects of Tubing Type, Formulation, and Postpumping Agitation on
           Nanoparticle and Microparticle Formation in Intravenous Immunoglobulin
           Solutions Processed With a Peristaltic Filling Pump
    • Abstract: Publication date: January 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 1Author(s): Cheng Her, John F. CarpenterAbstractThe main goal of this study was to use state-of-the-art instruments for nanoparticle (nanoparticle tracking analysis and resonant mass measurement) and microparticle counting (flow imaging) to assess the effects of peristaltic filling pump operation on particle formation in formulations of intravenous immunoglobulin. Microparticle levels were also measured with light obscuration. Postpumping agitation was studied as an accelerated degradation method, 3 different commercial peristaltic tubing types were tested, and the effects of formulation pH and inclusion of polysorbate 80 were determined. Overall, the results documented that nanoparticle measurements, as well as microparticle determinations with flow imaging, were essential to gain rigorous insights into impacts of processing and formulation parameters on pumping- and agitation-induced particle formation. In addition, light obscuration was a relatively insensitive method and failed to detect large increases in protein particles caused by pumping and postpumping agitation. Formulation studies showed that the presence of polysorbate 80 or increasing protein colloidal stability with appropriate choice of buffer generally reduced particle formation. The results highlight the need for filling pump assessments in formulation development studies. Combining such assessments with appropriate analytical methods should help assure that particle levels are controlled during filling pump operation and that the highest quality products are manufactured.
       
  • Monoclonal Antibody Dimers Induced by Low pH, Heat, or Light Exposure Are
           Not Immunogenic Upon Subcutaneous Administration in a Mouse Model
    • Abstract: Publication date: January 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 1Author(s): Grzegorz Kijanka, Jared S. Bee, Mark A. Schenerman, Samuel A. Korman, Yuling Wu, Bram Slütter, Wim JiskootAbstractThe presence of protein aggregates is commonly believed to be an important risk factor for immunogenicity of therapeutic proteins. Among all types of aggregates, dimers are relatively abundant in most commercialized monoclonal antibody (mAb) products. The aim of this study was to investigate the immunogenicity of artificially created mAb dimers relative to that of unstressed and stressed mAb monomers. A monoclonal murine IgG1 (mIgG1) antibody was exposed to low pH, elevated temperature, or UV irradiation to induce dimerization. Dimers and monomers were purified via size-exclusion chromatography. Physicochemical analysis revealed that upon all stress conditions, new deamidation or oxidation or both of amino acids occurred. Nevertheless, the secondary and tertiary structures of all obtained dimers were similar to those of unstressed mIgG1. Isolated dimers were administered subcutaneously in Balb/c mice, and development of antidrug antibodies and accumulation of follicular T helper cells in draining lymph nodes and spleens were determined. None of the tested dimers or stressed monomers were found to be more immunogenic than the unstressed control in our mouse model. In conclusion, both dimers and monomers generated by using 3 different stress factors have a low immunogenicity similar to that of the unstressed monomers.
       
  • Near-Infrared Spectroscopy to Determine Residual Moisture in Freeze-Dried
           Products: Model Generation by Statistical Design of Experiments
    • Abstract: Publication date: January 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 1Author(s): Matthieu Clavaud, Carmen Lema-Martinez, Yves Roggo, Michael Bigalke, Aurélie Guillemain, Philippe Hubert, Eric Ziemons, Andrea AllmendingerAbstractMoisture content (MC) is a critical quality attribute of lyophilized biopharmaceuticals and can be determined by near-infrared (NIR) spectroscopy as nondestructive alternative to Karl-Fischer titration. In this study, we create NIR models to determine MC in mAb lyophilisates by use of statistical design of experiments (DoE) and multivariate data analysis. We varied the composition of the formulation as well as lyophilization parameters covering a large range of representative conditions, which is commonly referred to as “robustness testing” according to quality-by-design concepts. We applied principles of chemometrics with partial least squares and principal component analysis. The NIR model excluded samples with complete collapse and MC> 6%. The 2 main components in the principal component analysis were MC (91%) and protein:sugar ratio (6%). The third component amounted to only 3% and remained unspecified but may include variations in process parameters and cake structure. In contrast to traditional approaches for NIR model creation, the DoE-based model can be used to monitor MC during drug product development work including scale-up, and transfer without the need to update the NIR model if protein:sugar ratio and MC stays within the tested limits and cake structure remains macroscopically intact. The use of the DoE approach and multivariate data analysis ensures product consistency and improves understanding of the manufacturing process.
       
  • A Novel Photoreactive Excipient to Probe Peptide-Matrix Interactions in
           Lyophilized Solids
    • Abstract: Publication date: January 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 1Author(s): Yuan Chen, Elizabeth M. ToppAbstractExcipients used in lyophilized protein drug products are often selected by a trial-and-error method, in part, because the analytical methods used to detect protein-excipient interactions in lyophilized solids are limited. In this study, photolytic labeling was used to probe interactions between salmon calcitonin (sCT) and excipients in lyophilized solids. Two diazirine-derived photo-excipients, photo-leucine (pLeu) and photo-glucosamine (pGlcN), were incorporated into lyophilized solids containing sCT, together with an unlabeled excipient (sucrose or histidine) at prelyophilization pH values from 6 to 9.9. Commercially available pLeu was selected as an ionizable photo-excipient and amino acid analog, while pGlcN was synthesized as an analog of sugar-based excipients. Photolytic labeling was induced by exposing the solids to UV light (365 nm, 30-60 min), and the resulting products were identified and quantified with liquid-chromatography mass spectrometry. The distribution of photo-reaction products was affected by the photoreactive reagent used, the type of unlabeled excipient, and the solution pH before lyophilization. When other components of the solid were identical, the extent and sites of labeling on sCT were different for pGlcN and pLeu. The results suggest that ionizable and nonionizable excipients interact differently with sCT in lyophilized solids and that photo-excipients can be used to map these interactions.
       
  • Effects of Glycan Structure on the Stability and Receptor Binding of an
           IgG4-Fc
    • Abstract: Publication date: January 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 1Author(s): Huan Kang, Nicholas R. Larson, Derek R. White, C. Russell Middaugh, Thomas Tolbert, Christian SchöneichAbstractA series of well-defined N-glycosylated IgG4-Fc variants were utilized to investigate the effect of glycan structure on their physicochemical properties (conformational stability and photostability) and interactions with an Fc γ receptor IIIA (FcγRIIIA). High mannose (HM, GlcNAc2Man(8+n) [n = 0-4]), Man5 (GlcNAc2Man5), GlcNAc1, and N297Q IgG4-Fc were prepared in good quality. The physical stability of these IgG4-Fc variants was examined with differential scanning calorimetry and intrinsic fluorescence spectroscopy. Photostability was assessed after photoirradiation between 295 and 340 nm (λ max = 305 nm), and HPLC-MS/MS analysis of specific products was performed. The size of glycans at Asn297 affects the yields of light-induced Tyr side-chain fragmentation products, where the yields decreased in the following order: N297Q> GlcNAc1> Man5> HM. These yields correlate with the thermal stability of the glycoforms. The HM and Man5 glycoforms display increased affinity for FcγRIIIA by at least 14.7-fold compared with GlcNAc1 IgG4-Fc. The affinities measured for the HM and Man5 IgG4-Fc (0.39-0.52 μM) are similar to those measured for fucosylated IgG1. Dependent on the mechanisms of action of IgG4 therapeutics, such glycoforms may need to be carefully monitored. The nonglycosylated N297Q IgG4-Fc did not present measurable affinity to FcγRIIIA.
       
  • Mechanical Shock During Shipping of Medications: The Roles of Packaging
           and Transportation Vendors
    • Abstract: Publication date: January 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 1Author(s): Sarah Dill, Kyle Brees, Andrew Stahly, Elizabeth Cheng, John Carpenter, Liron CaplanAbstractProtein-based medications are expensive and susceptible to damage from mechanical shock, which may occur during shipping to patients from pharmacies. Our objectives were to evaluate the performance of 2 packaging systems and to describe the mechanical shock that occurs during shipments. The packaging systems evaluated were boxes containing expanded polystyrene (EPS) or soft lint-like foam, prepared with and without polymer cooling packs. In laboratory-based studies, accelerometers measured g forces in boxes dropped from varying heights. Transportation studies evaluated the EPS cooler when sent to locations via 2 vendors. The relationship between drop height and maximum force vector was approximately linear for drop heights of 7.5″ through 30″, with lower magnitude forces at 45″ and 60″. Soft foam reduced force by 9.8 g on average compared to EPS (p < 0.009). The presence of polymer packs mitigated forces; frozen packs reduced forces by 28 g versus thawed packs (p < 0.001). Transportation experiments demonstrated most impacts were in the low (10-24 g) and low-medium (25-49 g) range (95% of all impacts). There was no difference between impacts during shipments with the vendors, and there was no correlation between distance traveled and number of impacts. Overall, mechanical shock during shipping is both prevalent and contingent upon the packing materials used.
       
  • Unique Impacts of Methionine Oxidation, Tryptophan Oxidation, and
           Asparagine Deamidation on Antibody Stability and Aggregation
    • Abstract: Publication date: January 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 1Author(s): Magfur E. Alam, Thomas R. Slaney, Lina Wu, Tapan K. Das, Sambit Kar, Gregory V. Barnett, Anthony Leone, Peter M. TessierAbstractMonoclonal antibodies are attractive therapeutic agents because of their impressive biological activities and favorable biophysical properties. Nevertheless, antibodies are susceptible to various types of chemical modifications, and the impact of such modifications on antibody physical stability and aggregation remains understudied. Here, we report a systematic analysis of the impact of methionine oxidation, tryptophan oxidation, and asparagine deamidation on antibody conformational and colloidal stability, hydrophobicity, solubility, and aggregation. Interestingly, we find little correlation between the impact of these chemical modifications on antibody conformational stability and aggregation. Methionine oxidation leads to significant reductions in antibody conformational stability while having little impact on antibody aggregation except at extreme conditions (low pH and elevated temperature). Conversely, tryptophan oxidation and asparagine deamidation have little impact on antibody conformational stability while promoting aggregation at a wide range of solution conditions, and the aggregation mechanisms appear linked to unique types of reducible and nonreducible covalent crosslinks and, in some cases, to increased levels of attractive colloidal interactions. These findings highlight that even related types of chemical modifications can lead to dissimilar antibody aggregation mechanisms, and evaluating these findings for additional antibodies will be important for improving the systematic generation of antibodies with high chemical and physical stability.
       
  • Novel High-Throughput Assay for Polysorbate Quantification in
           Biopharmaceutical Products by Using the Fluorescent Dye DiI
    • Abstract: Publication date: January 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 1Author(s): Ariadna Martos, Michelle Berger, Wendelin Kranz, Anna Spanopoulou, Tim Menzen, Wolfgang Friess, Klaus Wuchner, Andrea HaweAbstractPolysorbates (PSs) are the most common surfactants in therapeutic protein formulations, and it is crucial to monitor their concentration along the life cycle of biopharmaceuticals. We developed a simple multi-well plate fluorescence-based assay for the rapid determination of PS20 and PS80 content in biopharmaceutical products. The method is based on the detection of the fluorescence emission intensity of the fluorescent dye 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate in the presence of PSs at concentrations below their critical micelle concentration. This method can be applied for PS content determination in protein formulations (≤100 mg/mL) without the need of a previous protein removal step. The 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate assay implemented in multi-well plate format is suitable for high-throughput concentration screening. It has a linear range from 0.00020% to 0.0025% (w/v) PS20, and the limits of detection and quantification were 0.00020% and 0.00055% (w/v), respectively. This assay is markedly more selective and shows no or lower interferences due to hydrophobic components (e.g., silicone oil) potentially present in finished products than the fluorescence micelle assay based on N-phenyl-1-naphthylamine. It also provides comparable results for the PS content in liquid chromatography with charged aerosol detection analysis with protein removal, providing a fast alternative.
       
  • Characterization of Subvisible Particles in Biotherapeutic Prefilled
           Syringes: The Role of Polysorbate and Protein on the Formation of Silicone
           Oil and Protein Subvisible Particles After Drop Shock
    • Abstract: Publication date: January 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 1Author(s): Nancy Jiao, Gregory V. Barnett, Twinkle R. Christian, Linda O. Narhi, Nathan H. Joh, Marisa K. Joubert, Shawn CaoAbstractSubvisible particles (SbVPs) are a critical quality attribute for biotherapeutics. Particle content in prefilled syringes (PFSs) of a biotherapeutic can include protein particles and silicone oil particles (SiOP). Here, a real-world protein therapeutic PFS shows that although polysorbate is effective in preventing protein particle formation, it also leads to the formation of SiOP. PFSs of protein and buffer formulations in the presence and absence of polysorbate are subjected to a drop shock to generate SbVP and the effect of polysorbate and protein in generating SbVP is investigated. Particle characterization by light obscuration and flow imaging shows that polysorbate prevents protein particle formation as intended, but the presence of polysorbate substantially increases the formation of SiOP. The protein itself also acts as a surfactant and leads to increased SiOP, but to a lesser degree compared to polysorbate. In a separate companion study by Joh et al., the risk of immunogenicity was assessed using in vivo and in vitro models. Flow imaging distinguishes between SiOP and protein particles and enables risk assessment of the natures of different SbVP in PFSs.
       
  • A Chemometric Approach Toward Predicting the Relative Aggregation
           Propensity: Aβ(1-42)
    • Abstract: Publication date: January 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 1Author(s): Nathaniel J. Zbacnik, Charles S. Henry, Mark Cornell ManningAbstractA number of algorithms have been developed to predict the aggregation propensity of peptides and proteins, but virtually none have the ability to provide sequence-specific information on what physicochemical properties are most important in altering aggregation propensity. In this study, a chemometric approach using reduced amino acid properties is used to examine the aggregation behavior of a highly amyloidogenic peptide, Aβ(1-42). Specific residues are identified as being critical to the aggregation process. At each of these positions, the important physicochemical properties are identified that would either accelerate or inhibit fibril formation.
       
  • Automatic Identification of the Stress Sources of Protein Aggregates Using
           Flow Imaging Microscopy Images
    • Abstract: Publication date: January 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 1Author(s): Arni Gambe-Gilbuena, Yuriko Shibano, Elena Krayukhina, Tetsuo Torisu, Susumu UchiyamaAbstractA novel approach to identify 5 types of simulated stresses that induce protein aggregation in prefilled syringe–type biopharmaceuticals was developed. Principal components analyses of texture metrics extracted from flow imaging microscopy images were used to define subgroups of particles. Supervised machine learning methods, including convolutional neural networks, were used to train classifiers to identify subgroup membership of constituent particles to generate distribution profiles. The applicability of the stress-specific signatures for distinguishing stress source types was verified. The high classification efficiencies (100%) precipitated the collection of data from more than 20 independent experiments to train support vector machines, k-nearest neighbors, and ensemble classifiers. The performances of the trained classifiers were validated. High classification efficiencies for friability (80%-100%) and heating at 90°C (85%-100%) are indicative of high reliability of these methods for stress-stability assays while extreme variations in freeze-thawing (2%-100%) and heating at 60°C (2.25%-98.25%) indicate the unpredictability of particle composition profiles for these forced degradation conditions. We also developed subvisible particle classifiers using convolutional neural network to automatically identify silicone oil droplets, air bubbles, and protein aggregates. The developed classifiers will contribute to mitigating aggregation in biopharmaceuticals via the identification of stress sources.
       
  • Accelerated Aggregation Studies of Monoclonal Antibodies: Considerations
           for Storage Stability
    • Abstract: Publication date: January 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 1Author(s): Ruben Wälchli, Pieter-Jan Vermeire, Jan Massant, Paolo ArosioAbstractAggregation of mAbs is a crucial concern with respect to their safety and efficacy. Among the various properties of protein aggregates, it is emerging that their size can potentially impact their immunogenicity. Therefore, stability studies of antibody formulations should not only evaluate the rate of monomer loss but also determine the size distribution of the protein aggregates, which in turn depends on the aggregation mechanism. Here, we study the aggregation behavior of different formulations of 2 monoclonal immunoglobulins (IgGs) in the temperature range from 5°C to 50°C over 52 weeks of storage. We show that the aggregation kinetics of both antibodies follow non-Arrhenius behavior and that the aggregation mechanisms change between 40°C and 5°C, leading to different types of aggregates. Specifically, for a given monomer conversion, dimer formation dominates at low temperatures, while larger aggregates are formed at higher temperatures. We further show that the stability ranking of different molecules as well as of different formulations is drastically different at 40°C and 5°C while it correlates better between 30°C and 5°C. Our findings have implications for the level of information provided by accelerated aggregation studies with respect to protein stability under storage conditions.
       
  • Orthogonal Techniques to Study the Effect of pH, Sucrose, and Arginine
           Salts on Monoclonal Antibody Physical Stability and Aggregation During
           Long-Term Storage
    • Abstract: Publication date: January 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 1Author(s): Hristo L. Svilenov, Alina Kulakova, Matja Zalar, Alexander P. Golovanov, Pernille Harris, Gerhard WinterAbstractUnderstanding the effects of additives on therapeutic protein stability is of paramount importance for obtaining stable formulations. In this work, we apply several high- and medium-throughput methods to study the physical stability of a model monoclonal antibody at pH 5.0 and 6.5 in the presence of sucrose, arginine hydrochloride, and arginine glutamate. In low ionic strength buffer, the addition of salts reduces the antibody colloidal and thermal stability, attributed to screening of electrostatic interactions. The presence of glutamate ion in the arginine salt partially reduces the damaging effect of ionic strength increase. The addition of 280 mM sucrose shifts the thermal protein unfolding to a higher temperature. Arginine salts in the used concentration reduce the relative monomer yield after refolding from urea, whereas sucrose has a favorable effect on antibody refolding. In addition, we show 12-month long-term stability data and observe correlations between thermal protein stability, relative monomer yield after refolding, and monomer loss during storage. The monomer loss during storage is related to protein aggregation and formation of subvisible particles in some of the formulations. This study shows that the effect of commonly used additives on the long-term antibody physical stability can be predicted using orthogonal biophysical measurements.
       
  • Fcγ Receptor Activation by Human Monoclonal Antibody Aggregates
    • Abstract: Publication date: January 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 1Author(s): Minoru Tada, Michihiko Aoyama, Akiko Ishii-WatabeAbstractProtein aggregates are a potential risk factor for immunogenicity. The measurement, characterization, and control of protein aggregates in drug products are indispensable for the development of biopharmaceuticals, including therapeutic mAbs. In this study, Fcγ receptor (FcγR)-expressing reporter cell lines were used to analyze the FcγR-activation properties of mAb aggregates. Comparison of aggregates of mAbs harboring different IgG subclasses revealed that the FcγR-activation profiles of the mAb aggregates were dependent on IgG subclass. In addition, aggregates of Fc-engineered mAb with enhanced FcγR-activation properties exhibited stronger activation of FcγRs than was observed in the wild-type aggregates, whereas aggregates of Fc-engineered mAb with decreased FcγR-activation properties showed reduced activation. These results suggest that FcγR activation by mAb aggregates depends greatly on the Fc functions of the native (nonaggregated) mAbs. We also showed that aggregates of mAbs smaller than 1 μm in size have the potential to directly activate FcγRs. Unintended immune cell activation can be induced on account of FcγR activation by mAb aggregates and such FcγR activation may contribute to immunogenicity, and therefore, analysis of the FcγR-activation properties of mAb aggregates using FcγR-expressing reporter cell lines is a promising approach for the characterization of mAb aggregates.
       
  • Control of Antibody Impurities Induced by Riboflavin in Culture Media
           During Production
    • Abstract: Publication date: January 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 1Author(s): Alison Wallace, Stephen Trimble, John F. Valliere-Douglass, Martin Allen, Catherine Eakin, Alain Balland, Pranhitha Reddy, Michael J. TreuheitAbstractDuring the manufacturing of protein biologics, product variability during cell culture production and harvest needs to be actively controlled and monitored to maintain acceptable product quality. To a large degree, variants that have previously been described are covalent in nature and are easily analyzed by a variety of techniques. Here, we describe a noncovalent post translational modification of recombinantly expressed antibodies, containing variable domain tryptophans, that are exposed to culture media components and ambient laboratory light. The modified species, designated as conformer, can be monitored by hydrophobic interaction chromatography and often exhibits reduced potency. We studied conformer formation and identified key elements driving its accelerated growth using an IgG2 monoclonal antibody. Conformer is a result of a noncovalent interaction of the antibody with riboflavin, an essential vitamin added to many production cell culture formulations. Chemical and physical factors that influence the impact of riboflavin are identified, and methods for process control of this product quality attribute are addressed in order to prevent loss of antibody potency and potential safety issues. Identifying therapeutic antibody drug candidates with the potential to form conformers can be performed early in development to avoid this undesirable product quality propensity.
       
  • Subvisible Particles in IVIg Formulations Activate Complement in Human
           Serum
    • Abstract: Publication date: January 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 1Author(s): Carly F. Chisholm, William Behnke, Yekaterina Pokhilchuk, Ashley A. Frazer-Abel, Theodore W. RandolphAbstractWhen administered intravenously, various particles and nanomedicines activate complement, potentially leading to infusion reactions and other adverse drug reactions. Particles form within formulations of therapeutic proteins due to stresses incurred during shipping, handling, and administration to patients. In this study, IVIg solutions were stored in multiple types of vials and prefilled syringes and exposed to agitation and freeze-thaw stresses to generate particles. The stressed samples were added to human serum to determine whether these particles activated complement. Subvisible IVIg particles ranging in size between 2 and 10 microns activated complement in a fashion that was linearly dependent on the number of particles dosed, whereas little correlation was found between doses of larger particles (>10 microns) and complement activation. Activation of complement by subvisible particles of IVIg followed the alternative pathway, as shown by the release of complement cascade factor Bb and the production of the anaphylatoxins C3a and C5a without generation of C4a. The number and the morphology of subvisible particles formed depended on the applied stress, formulation, and on the container material. But the capacity of the 2- to 10-micron-sized particles to activate complement in human serum appeared to depend only on particle concentration.
       
  • Identification of Formaldehyde-Induced Modifications in Diphtheria Toxin
    • Abstract: Publication date: January 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 1Author(s): Bernard Metz, Thomas Michiels, Joost Uittenbogaard, Maarten Danial, Wichard Tilstra, Hugo D. Meiring, Wim E. Hennink, Daan J.A. Crommelin, Gideon F.A. Kersten, Wim JiskootAbstractDiphtheria toxoid is produced by detoxification of diphtheria toxin with formaldehyde. This study was performed to elucidate the chemical nature and location of formaldehyde-induced modifications in diphtheria toxoid. Diphtheria toxin was chemically modified using 4 different reactions with the following reagents: (1) formaldehyde and NaCNBH3, (2) formaldehyde, (3) formaldehyde and NaCNBH3 followed by formaldehyde and glycine, and (4) formaldehyde and glycine. The modifications were studied by SDS-PAGE, primary amino group determination, and liquid chromatography–electrospray mass spectrometry of chymotryptic digests. Reaction 1 resulted in quantitative dimethylation of all lysine residues. Reaction 2 caused intramolecular cross-links, including the NAD+-binding cavity and the receptor-binding site. Moreover, A fragments and B fragments were cross-linked by formaldehyde on part of the diphtheria toxoid molecules. Reaction 3 resulted in formaldehyde-glycine attachments, including in shielded areas of the protein. The detoxification reaction typically used for vaccine preparation (reaction 4) resulted in a combination of intramolecular cross-links and formaldehyde-glycine attachments. Both the NAD+-binding cavity and the receptor-binding site of diphtheria toxin were chemically modified. Although CD4+ T-cell epitopes were affected to some extent, one universal CD4+ T-cell epitope remained almost completely unaltered by the treatment with formaldehyde and glycine.
       
  • Evaluation of Crystal Zenith Microtiter Plates for High-Throughput
           Formulation Screening
    • Abstract: Publication date: January 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 1Author(s): J. Alaina Floyd, Jeremy M. Shaver, Alison J. Gillespie, Unjy Park, Richard S. Rogers, Nancy S. Nightlinger, Yuko Ogata, Jeffrey J. James, Bruce A. KerwinAbstractFormulation screening for biotherapeutics can cover a vast array of excipients and stress conditions. These studies consume quantities of limited material and, with higher concentrated therapeutics, more material is needed. Here, we evaluate the use of crystal zenith (CZ) microtiter plates in conjunction with FluoroTec-coated butyl rubber mats as a small-volume, high-throughput system for formulation stability studies. The system was studied for evaporation, edge effects, and stability with comparisons to type 1 glass and CZ vials for multiple antibodies and formulations. Evaporation was minimal at 4°C and could be reduced at elevated temperatures using sealed, mylar bags. Edge effects were not observed until 12 weeks at 40°C. The overall stability ranking as measured by the rate of change in high molecular weight and percent main peak species was comparable across both vials and plates at 4°C and 40°C out to 12 weeks. Product quality attributes as measured by the multi-attribute method were also comparable across all containers for each molecule formulation. A potential difference was measured for subvisible particle analysis, with the plates measuring lower particle counts than the vials. Overall, CZ plates are a viable alternative to traditional vials for small-volume, high-throughput formulation stability screening studies.
       
  • Technical Capabilities and Limitations of Optical Spectroscopy and
           Calorimetry Using Water-Miscible Solvents: The Case of Dimethyl Sulfoxide,
           Acetonitrile, and 1,4-Dioxane
    • Abstract: Publication date: January 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 1Author(s): Atsushi Hirano, Satoru Nagatoishi, Momoyo Wada, Kouhei Tsumoto, Karl N. Maluf, Tsutomu ArakawaAbstractIn drug development, water-miscible solvents are commonly used to dissolve drug substances. Typical routine procedures in drug development include dilution of the stock drug solution into an aqueous solution containing target macromolecules for drug binding assays. However, water-miscible solvents impose some technical limitations on the assays on account of their light absorption and heat capacity. Here, we examined the effects of the dilution of 3 water-miscible solvents, that is, dimethyl sulfoxide, acetonitrile, and 1,4-dioxane, on the baseline stability and signal/noise ratio in circular dichroism spectroscopy, isothermal titration calorimetry, and differential scanning calorimetry. Dimethyl sulfoxide and 1,4-dioxane affect the signal/noise ratio of circular dichroism spectra at typically used concentrations due to their light absorbance. The water-miscible solvents generate interfering signals in the isothermal titration calorimetry due to their mixing heat. They show negative or positive slope in the differential scanning calorimetry. Such interfering effects of the solvents are reduced by appropriate dilution according to the analytical techniques. Because the water-miscible solvents have solubilization capacity for alkyl chain moieties and aromatic moieties of chemicals, drug substances containing these moieties can be dissolved into the solvents and then subjected to the analyses to examine their interactions with target proteins after appropriate dilution of the drug solutions.
       
  • Assessment of the Injection Performance of a Tapered Needle for Use in
           Prefilled Biopharmaceutical Products
    • Abstract: Publication date: January 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 1Author(s): Elena Krayukhina, Ayano Fukuhara, Susumu UchiyamaAbstractThe design of injection devices, including prefilled syringes (PFSs) and autoinjectors, requires an understanding of the optimization of injection conditions. The injection of highly concentrated biopharmaceuticals can lead to exceptionally high injection forces, due to their high viscosity. To overcome this challenge, a tapered needle has been recently developed by Terumo Corporation. In the present study, we measured the injection forces in PFSs equipped with 24G-29G tapered needle (29G TNN), 27G thin-wall needle (27G TW), and 29G TW using several model and pharmaceutical protein solutions. The injection forces measured in the 29G TNN PFSs were lower than those in 29G TW for all solutions, similar to those in 27G TW PFSs for Newtonian solutions, and were lower than those in the 27G TW PFSs for non-Newtonian solutions which demonstrated shear-thinning behavior. No significant changes in aggregates or micron-size particle concentrations were observed upon injection, regardless of the needle type. Mathematical modeling supported the experimental findings that under similar flow rate conditions injection pressure in a tapered needle is lower than that in a cylindrical needle. Our results indicate that there are advantages of using tapered needles for the injection of biopharmaceutical formulations particularly those showing shear-thinning behavior.
       
  • Taking Subvisible Particle Quantitation to the Limit: Uncertainties and
           Statistical Challenges With Ophthalmic Products for Intravitreal Injection
           
    • Abstract: Publication date: January 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 1Author(s): Marina Gühlke, Johanna Hecht, Armin Böhrer, Andrea Hawe, Felix Nikels, Patrick Garidel, Tim MenzenAbstractSubvisible particles are a critical quality attribute of pharmaceutical products. The reliability of particle quantitation increases with the number of particles in the analyzed sample volume. However, for analyses of low-volume drug products, such as ophthalmic products for intravitreal injection or biopharmaceuticals in general, sample volumes as small as possible should be used to avoid pooling and consequently, the contamination with foreign particles. The aim of our study was to evaluate the variability of particle concentrations obtained by light obscuration measurements to define the minimum required analyzed sample volume to achieve statistically meaningful results by using conditions that are practically feasible. Statistical evaluation suggests that for particle concentrations close to a predefined limit, large sample volumes (a multiple of typical intravitreal product volumes) would be required for a high probability to correctly classify samples with respect to the predefined limit. Below a minimum analyzed volume, even a measurement result of 0 particles does not allow to conclude compliance with the respective particle concentration limit with sufficient certainty. A small analyzed volume could be justified as long as the measurement uncertainty remains acceptable compared with the predefined limit.
       
  • Trehalose or Sucrose: Which of the Two Should be Used for Stabilizing
           Proteins in the Solid State' A Dilemma Investigated by In Situ
           Micro-Raman and Dielectric Relaxation Spectroscopies During and After
           Freeze-Drying
    • Abstract: Publication date: January 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 1Author(s): Tatiana Starciuc, Benjamin Malfait, Florence Danede, Laurent Paccou, Yannick Guinet, Natalia T. Correia, Alain HedouxAbstractThe bioprotective properties of 2 disaccharides (sucrose and trehalose) were analyzed during the freeze-drying (FD) process and at the end of the process, to better understand the stabilization mechanisms of proteins in the solid state. In situ Raman investigations, performed during the FD process, have revealed that sucrose was more efficient than trehalose for preserving the secondary structure of lysozyme during FD, especially during the primary drying stage. The lower bioprotective effect of trehalose was interpreted as a consequence of a stronger affinity of this disaccharide to water, responsible for a severe phase separation phenomenon during the freezing stage. Dielectric spectroscopy investigations on the freeze-dried state of protein formulations have shown the capabilities of trehalose assisted by residual water to reduce the molecular mobility of the vitreous matrix, suggesting that trehalose is more efficient to preserve the protein structure during long-term storage.
       
  • Improving Diameter Accuracy for Dynamic Imaging Microscopy for Different
           Particle Types
    • Abstract: Publication date: January 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 1Author(s): Richard Ernest Cavicchi, Dean C. Ripple, Bioprocess Measurements GroupAbstractDynamic imaging analysis instruments are used for sizing particles of different types that might appear in a biopharmaceutical. These instruments are calibrated using polystyrene latex microspheres in water, which is a significantly different system than the typical particles imaged in a formulation. We show how the instruments, when reporting an equivalent diameter, set a threshold for image processing and then apply a built-in correction to account for fuzzy boundary effects. We investigate the degree to which the threshold value and built-in correction influences the size, and ultimately particle size distribution, that the instrument reports on other particle types. Size corrections for a dynamic imaging system in a typical optical configuration were determined by comparison of equivalent image diameters with diameters from Brownian motion tracking of particles. A variety of particles were characterized: aggregates made from a monoclonal antibody available as reference material RM 8671 from the National Institute of Standards and Technology, bovine serum albumin aggregates, silicone oil droplets, polystyrene microspheres, and ethylene tetrafluoroethylene particles, a protein aggregate simulant (National Institute of Standards and Technology reference material RM 8634). The results show that the protein aggregates and ethylene tetrafluoroethylene are very similar to one another but quite different from the polystyrene calibration spheres. This points the way to developing new correction factors and calibration procedures based on particle type.
       
  • Effect of Aluminum Adjuvant and Preservatives on Structural Integrity and
           Physicochemical Stability Profiles of Three Recombinant Subunit Rotavirus
           Vaccine Antigens
    • Abstract: Publication date: January 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 1Author(s): Sanjeev Agarwal, John M. Hickey, David McAdams, Jessica A. White, Robert Sitrin, Lakshmi Khandke, Stanley Cryz, Sangeeta B. Joshi, David B. VolkinAbstractA nonreplicating rotavirus vaccine (NRRV) containing 3 recombinant fusion proteins adsorbed to aluminum adjuvant (Alhydrogel [AH]) is currently in clinical trials. The compatibility and stability of monovalent NRRV antigen with key components of a multidose vaccine formulation were examined using physicochemical and immunochemical methods. The extent and strength of antigen-adjuvant binding were diminished by increasing phosphate concentration, and acceptable levels were identified along with alternate buffering agents. Addition of the preservative thimerosal destabilized AH-adsorbed P2-VP8-P[8] as measured by differential scanning calorimetry. Over 3 months at 4°C, AH-adsorbed P2-VP8-P[8] was stable, whereas at 25°C and 37°C, instability was observed which was greatly accelerated by thimerosal addition. Loss of antibody binding (enzyme-linked immunosorbent assay) correlated with loss of structural integrity (differential scanning calorimetry, fluorescence spectroscopy) with concomitant nonnative disulfide bond formation (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and Asn deamidation (liquid chromatography -mass spectrometry peptide mapping). An alternative preservative (2-phenoxyethanol) showed similar antigen destabilization. Due to limited availability, only key assays were performed with monovalent P2-VP8-P[4] and P2-VP8-P[6] AH-adsorbed antigens, and varying levels of preservative incompatibility were observed. In summary, monovalent AH-adsorbed NRRV antigens stored at 4°C showed good stability without preservatives; however, future formulation development efforts are required to prepare a stable, preservative-containing, multidose NRRV formulation.
       
  • Considerations on Protein Stability During Freezing and Its Impact on the
           Freeze-Drying Cycle: A Design Space Approach
    • Abstract: Publication date: January 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 1Author(s): Andrea Arsiccio, Paolo Giorsello, Livio Marenco, Roberto PisanoAbstractFreezing is widely used during the manufacturing process of protein-based therapeutics, but it may result in undesired loss of biological activity. Many variables come into play during freezing that could adversely affect protein stability, creating a complex landscape of interrelated effects. The current approach to the selection of freezing conditions is however nonsystematic, resulting in poor process control. Here we show how mathematical models, and a design space approach, can guide the selection of the optimal freezing protocol, focusing on protein stability. Two opposite scenarios are identified, suggesting that the ice-water interface is the dominant cause of denaturation for proteins with high bulk stability, while the duration of the freezing process itself is the key parameter to be controlled for proteins that are susceptible to cold denaturation. Experimental data for lactate dehydrogenase and myoglobin as model proteins support the model results, with a slow freezing rate being optimal for lactate dehydrogenase and the opposite being true for myoglobin. A possible application of the calculated design space to the freezing and freeze-drying of biopharmaceuticals is finally described, and some considerations on process efficiency are discussed as well.
       
  • Evaluation of Nanoparticle Tracking Analysis for the Detection of
           Rod-Shaped Particles and Protein Aggregates
    • Abstract: Publication date: January 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 1Author(s): Brandon M. Hoover, Regina M. MurphyAbstractNanoparticle tracking analysis (NTA) is an important technique for measuring hydrodynamic size of globular biological particles including liposomes and viruses. Less attention has been paid to NTA of rod-like particles, despite their considerable interest. For example, amyloid fibrils and protofibrils are protein aggregates with rod-like morphology, diameters of 2-15 nm, and lengths from 50 nm to 1 μm, and linked to diseases including Alzheimer’s and Parkinson’s. We used NTA to measure the concentration and hydrodynamic size of gold nanorods (10 nm diameter, 35-250 nm length) and myosin (2 nm diameter, 160 nm length), as models of rod-like particles. Measured hydrodynamic diameters of gold nanorods were consistent with theoretical calculations, as long as particle concentration and solution conditions were controlled. Myosin monomers were invisible by NTA, but a small population of aggregates was detected. We combined NTA results with other light scattering data to gain insight into number and size distribution of protein solutions containing both monomer and aggregates. Finally, we demonstrated the utility of NTA and its limitations by characterizing aggregates of alpha-synuclein. Of note is the use of NTA to detect a change in morphology from compact to elongated by analyzing the ratio of hydrodynamic size to intensity.
       
  • Characterization of Native Reversible Self-Association of a Monoclonal
           Antibody Mediated by Fab-Fab Interaction
    • Abstract: Publication date: January 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 1Author(s): Lorenzo Gentiluomo, Dierk Roessner, Werner Streicher, Sujata Mahapatra, Pernille Harris, Wolfgang FrießAbstractThe native reversible self-association of monoclonal antibodies has been associated with high viscosity, liquid-liquid, and liquid-solid phase separation. We investigated the native reversible self-association of an IgG1, which exerts this association even at low protein concentrations, in detail to gain further understanding of this phenomenon by extensive characterization of the association as a function of multiple factors, namely pH, temperature, salt concentration, and protein concentration. The nature of the self-association of the full-length IgG1 as well as the corresponding Fab and Fc fragment was studied by viz. size exclusion chromatography combined with multiangle light scattering, batch dynamic and static light scattering, analytical ultracentrifugation, small angle X-ray scattering, asymmetric flow field flow fractionation coupled with multiangle light scattering, and intrinsic fluorescence. We rationalized the self-association as a combination of hydrophobic and electrostatic interactions driven by the Fab fragments. Finally, we investigated the long-term stability of the IgG1 molecule.
       
  • DEHP Nanodroplets Leached From Polyvinyl Chloride IV Bags Promote
           Aggregation of IVIG and Activate Complement in Human Serum
    • Abstract: Publication date: January 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 1Author(s): Jared R. Snell, Connor R. Monticello, Cheng Her, Emma L. Ross, Ashley A. Frazer-Abel, John F. Carpenter, Theodore W. RandolphAbstractConcerns regarding the impact of subvisible particulate impurities on the safety and efficacy of therapeutic protein products have led manufacturers to implement strategies to minimize protein aggregation and particle formation during manufacturing, storage, and shipping. However, once these products are released, manufacturers have limited control over product handling. In this work, we investigated the effect of di(2-ethylhexyl) phthalate (DEHP) nanodroplets generated in polyvinyl chloride (PVC) bags of intravenous (IV) saline on the stability and immunogenicity of IV immunoglobulin (IVIG) formulations. We showed that PVC IV bags containing saline can release DEHP droplets into the solution when agitated or transported using a pneumatic tube transportation system in a clinical setting. We next investigated the effects of emulsified DEHP nanodroplets on IVIG stability and immunogenicity. IVIG adsorbed strongly to DEHP nanodroplets, forming a monolayer. In addition, DEHP nanodroplets accelerated IVIG aggregation in agitated samples. The immunogenicity of DEHP nanodroplets and IVIG aggregates generated in these formulations were evaluated using an in vitro assay of complement activation in human serum. The results suggested DEHP nanodroplets shed from PVC IV bags could reduce protein stability and induce activation of the complement system, potentially contributing to adverse immune responses during the administration of therapeutic proteins.
       
  • Structure-Function Assessment and High-Throughput Quantification of
           Site-Specific Aspartate Isomerization in Monoclonal Antibody Using a Novel
           Analytical Tool Kit
    • Abstract: Publication date: January 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 1Author(s): Kaimeng Zhou, Xiang Cao, James Bautista, Zhi Chen, Neil Hershey, Richard Ludwig, Li Tao, Ming Zeng, Tapan K. DasAbstractIsomerization of surface-exposed aspartic acid (Asp) in the complementarity-determining regions of therapeutic proteins could potentially impact their target binding affinity because of the sensitive location, and often requires complex analytical tactics to understand its effect on structure-function and stability. Inaccurate quantitation of Asp-isomerized variants, especially the succinimide intermediate, presents major challenge in understanding Asp degradation kinetics, its stability, and consequently establishing a robust control strategy. As a practical solution to this problem, a comprehensive analytical tool kit has been developed, which provides a solution to fully characterize and accurately quantify the Asp-related product variants. The toolkit offers a combination of 2 steps, an ion-exchange chromatography method to separate and enrich the isomerized variants in the folded structure for structure-function evaluation and a novel focused peptide mapping method to quantify the individual complementarity-determining region isomerization components including the unmodified Asp, succinimide, and isoaspartate. This novel procedure allowed an accurate quantification of each Asp-related variant and a comprehensive assessment of the functional impact of Asp isomerization, which ultimately helped to establish an appropriate control strategy for this critical quality attribute.
       
  • Preformulation Characterization and Stability Assessments of Secretory IgA
           Monoclonal Antibodies as Potential Candidates for Passive Immunization by
           Oral Administration
    • Abstract: Publication date: January 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 1Author(s): Yue Hu, Ozan S. Kumru, Jian Xiong, Lorena R. Antunez, John Hickey, Yang Wang, Lisa Cavacini, Mark Klempner, Sangeeta B. Joshi, David B. VolkinAbstractEnterotoxigenic Escherichia coli (ETEC) is a major cause of diarrheal disease among children in developing countries, and there are no licensed vaccines to protect against ETEC. Passive immunization by oral delivery of ETEC-specific secretory IgAs (sIgAs) could potentially provide an alternative approach for protection in targeted populations. In this study, a series of physiochemical techniques and an in vitro gastric digestion model were used to characterize and compare key structural attributes and stability profiles of 3 anti–heat-labile enterotoxin mAbs (sIgA1, sIgA2, and IgG1 produced in CHO cells). The mAbs were evaluated in terms of primary structure, N-linked glycan profiles, size and aggregate content, relative apparent solubility, conformational stability, and in vitro antigen binding. Compared to IgG1 mAb, sIgA1 and sIgA2 mAbs showed increased sample heterogeneity, especially in terms of N-glycan composition and the presence of higher molecular weight species. The sIgA mAbs showed overall better physical stability and were more resistant to loss of antigen binding activity during incubation at low pH, 37°C with pepsin. These results are discussed in terms of future challenges to design stable, low-cost formulations of sIgA mAbs as an oral supplement for passive immunization to protect against enteric diseases in the developing world.
       
  • Characterizing and Minimizing Aggregation and Particle Formation of Three
           Recombinant Fusion-Protein Bulk Antigens for Use in a Candidate Trivalent
           Rotavirus Vaccine
    • Abstract: Publication date: January 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 1Author(s): Sanjeev Agarwal, Neha Sahni, John M. Hickey, George A. Robertson, Robert Sitrin, Stanley Cryz, Sangeeta B. Joshi, David B. VolkinAbstractIn a companion paper, the structural integrity, conformational stability, and degradation mechanisms of 3 recombinant fusion-protein antigens comprising a non-replicating rotavirus (NRRV) vaccine candidate (currently being evaluated in early-stage clinical trials) are described. In this work, we focus on the aggregation propensity of the 3 NRRV antigens coupled to formulation development studies to identify common frozen bulk candidate formulations. The P2-VP8-P[8] antigen was most susceptible to shaking and freeze-thaw–induced aggregation and particle formation. Each NRRV antigen formed aggregates with structurally altered protein (with exposed apolar regions and intermolecular β-sheet) and dimers containing a non-native disulfide bond. From excipient screening studies with P2-VP8-P[8], sugars or polyols (e.g., sucrose, trehalose, mannitol, sorbitol) and various detergents (e.g., Pluronic F-68, polysorbate 20 and 80, PEG-3350) were identified as stabilizers against aggregation. By combining promising additives, candidate bulk formulations were optimized to not only minimize agitation-induced aggregation, but also particle formation due to freeze-thaw stress of P2-VP8-P[8] antigen. Owing to limited material availability, stabilization of the P2-VP8-P[4] and P2-VP8-P[6] was confirmed with the lead candidate P2-VP8-P[8] formulations. The optimization of these bulk NRRV candidate formulations is discussed in the context of subsequent drug product formulations in the presence of aluminum adjuvants.
       
  • Recombinant Subunit Rotavirus Trivalent Vaccine Candidate: Physicochemical
           Comparisons and Stability Evaluations of Three Protein Antigens
    • Abstract: Publication date: January 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 1Author(s): Sanjeev Agarwal, John M. Hickey, Neha Sahni, Ronald T. Toth, George A. Robertson, Robert Sitrin, Stanley Cryz, Sangeeta B. Joshi, David B. VolkinAbstractAlthough live attenuated Rotavirus (RV) vaccines are available globally to provide protection against enteric RV disease, efficacy is substantially lower in low- to middle-income settings leading to interest in alternative vaccines. One promising candidate is a trivalent nonreplicating RV vaccine, comprising 3 truncated RV VP8 subunit proteins fused to the P2 CD4+ epitope from tetanus toxin (P2-VP8-P[4/6/8]). A wide variety of analytical techniques were used to compare the physicochemical properties of these 3 recombinant fusion proteins. Various environmental stresses were used to evaluate antigen stability and elucidate degradation pathways. P2-VP8-P[4] and P2-VP8-P[6] displayed similar physical stability profiles as function of pH and temperature while P2-VP8-P[8] was relatively more stable. Forced degradation studies revealed similar chemical stability profiles with Met1 most susceptible to oxidation, the single Cys residue (at position 173/172) forming intermolecular disulfide bonds (P2-VP8-P[6] was most susceptible), and Asn7 undergoing the highest levels of deamidation. These results are visualized in a structural model of the nonreplicating RV antigens. The establishment of key structural attributes of each antigen, along with corresponding stability-indicating methods, have been applied to vaccine formulation development efforts (see companion paper), and will be utilized in future analytical comparability assessments.
       
  • Shape Characterization of Subvisible Particles Using Dynamic Imaging
           Analysis
    • Abstract: Publication date: January 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 1Author(s): Roman Mathaes, Mark Cornell Manning, Gerhard Winter, Julia Engert, Glenn A. WilsonAbstractProtein aggregates and subvisible particles (SbvP), inherently present in all marketed protein drug products, have received increasing attention by health authorities. Dynamic imaging analysis was introduced to visualize SbvP and facilitate understanding of their origin. The educational United States Pharmacopeia chapter emphasizes that dynamic imaging analysis could be used for morphology measurements in the size range of 4-100 μm. However, adequate morphology characterization, as suggested in the United States Pharmacopeia proposed size range, remains challenging as nonspherical size standards are not commercially available. In this study, a homogenous and well-defined nonspherical particle standard was fabricated and used to investigate the capabilities of 2 dynamic imaging analysis systems (microflow imaging (MFI) and FlowCAM) to characterize SbvP shape in the size range of 2-10 μm. The actual aspect ratio of the SbvP was measured by scanning electron microscopy and compared to the results obtained by dynamic imaging analysis. The test procedure was used to assess the accuracy in determining the shape characteristics of the nonspherical particles. In general, dynamic imaging analysis showed decreasing accuracy in morphology characterization for 5 μm and 2 μm particles. The test procedure was also capable to compare and evaluate differences between the 2 dynamic imaging methods. The present study should help to define ranges of operation for dynamic imaging analysis systems.
       
  • Characterization of Protein Aggregates, Silicone Oil Droplets, and
           Protein-Silicone Interactions Using Imaging Flow Cytometry
    • Abstract: Publication date: January 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 1Author(s): Christine ProbstAbstractProtein therapeutic exposure to siliconized surfaces during manufacturing and storage has potential to induce protein aggregation or generate protein-silicone complexes that are potentially immunogenic. Consequently, assessing the stability and safety of protein therapeutics requires discrimination of protein and silicone oil particles and evaluation of protein–silicone oil interactions. Industry-established methods are challenged to distinguish protein aggregates from silicone oil droplets for particles smaller than 5 μm. In addition, emerging techniques for accessing particles in the sub-5 μm range are limited by low sampling volumes, narrow size ranges, complications such as clogging, and an inability to evaluate protein-silicone interactions. In this study, imaging flow cytometry was evaluated as a new method for discrimination of protein aggregates and silicone oil droplets, as well as for quantification of protein-silicone interactions. A simple and fast fluorescence labeling protocol using low concentrations of extrinsic dyes was developed. The results demonstrate that imaging flow cytometry can be used to discriminate fluorescently labeled protein aggregates and silicone oil droplets with greater than 95% accuracy, regardless of size, and protein–silicone oil interactions can be assessed qualitatively through inspection of image data or quantitatively using features extracted from the image data.
       
  • Characterization of Excipient Effects on Reversible Self-Association,
           Backbone Flexibility, and Solution Properties of an IgG1 Monoclonal
           Antibody at High Concentrations: Part 2
    • Abstract: Publication date: January 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 1Author(s): Yue Hu, Ronald T. Toth, Sangeeta B. Joshi, Reza Esfandiary, C. Russell Middaugh, David B. Volkin, David D. WeisAbstractIn this work, we continue to examine excipient effects on the reversible self-association (RSA) of 2 different IgG1 monoclonal antibodies (mAb-J and mAb-C). We characterize the RSA behavior of mAb-C which, similar to mAb-J (see Part 1), undergoes concentration-dependent RSA, but by a different molecular mechanism. Five additives that affect protein hydrophobic interactions to varying extents including a chaotropic salt (guanidine hydrochloride), a hydrophobic salt (trimethylphenylammonium iodide), an aromatic amino acid derivative (tryptophan amide hydrochloride), a kosmotropic salt (sodium sulfate, Na2SO4), and a less polar solvent (ethanol) were evaluated to determine their effects on the solution properties, molecular properties, and RSA of mAb-C at various protein concentrations. Four of the 5 additives examined demonstrated favorable effects on the pharmaceutical properties of high concentration mAb-C solutions (i.e., lower viscosity and weakened protein-protein interactions, PPIs) with a ranking order of guanidine hydrochloride> trimethylphenylammonium iodide> tryptophan amide hydrochloride> ethanol as measured by various biophysical techniques. Conversely, addition of Na2SO4 resulted in less desirable solution properties and enhanced PPIs. The effect of these 5 additives on mAb-C backbone dynamics were evaluated by hydrogen exchange-mass spectrometry (at high vs. low protein concentrations) to better understand their effects on the molecular sites of RSA in mAb-C.
       
  • Characterization of Excipient Effects on Reversible Self-Association,
           Backbone Flexibility, and Solution Properties of an IgG1 Monoclonal
           Antibody at High Concentrations: Part 1
    • Abstract: Publication date: January 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 1Author(s): Yue Hu, Jayant Arora, Sangeeta B. Joshi, Reza Esfandiary, C. Russell Middaugh, David D. Weis, David B. VolkinAbstractMany challenges limit the formulation of antibodies as high-concentration liquid dosage forms including elevated solution viscosity, decreased physical stability, and in some cases, liquid-liquid phase separation. In this work, an IgG1 monoclonal antibody (mAb-J), which undergoes concentration-dependent reversible self-association (RSA), is characterized in the presence of 4 amino acids (Arg, Lys, Asp, Glu) and NaCl using biophysical techniques and hydrogen exchange-mass spectrometry. The 5 additives disrupt RSA, prevent phase separation, and reduce solution viscosity to varying extents. These excipients also cause decreased turbidity, reduced average hydrodynamic diameter, and increased relative solubility of mAb-J in solution. The RSA disrupting efficacy of the positively charged amino acids is greater than either negatively charged amino acids or NaCl. As measured by hydrogen exchange-mass spectrometry, anionic excipients induced more alterations of mAb-J backbone dynamics at pH 6.0, and weak Fab-Fab interactions likely remained with the addition of either cationic or anionic excipients at high protein concentrations. Along with a companion paper examining a different mAb with a different molecular mechanism of RSA, these results are discussed in the context of various excipient strategies to disrupt protein-protein interactions to formulate mAbs at high protein concentrations with good stability profiles and favorable pharmaceutical properties for subcutaneous administration.
       
  • Time-Dependent Multi-Light-Source Image Classification Combined With
           Automated Multidimensional Protein Phase Diagram Construction for Protein
           Phase Behavior Analysis
    • Abstract: Publication date: January 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 1Author(s): Marieke E. Klijn, Jürgen HubbuchAbstractImage-based protein phase diagram analysis is key for understanding and exploiting protein phase behavior in the biopharmaceutical field. However, required data analysis has become a notorious time-consuming task since high-throughput screening approaches were implemented. A variety of computational tools have been developed to support analysis, but these tools primarily use end point visible light images. This study investigates the combined effect of end point and time-dependent image features obtained from cross-polarized and ultraviolet light features, supplementary to visible light, on protein phase diagram image classification. In addition, external validation was performed to evaluate the classification algorithm’s applicability to support protein phase diagram scoring. The predicted protein phase behavior classes were subsequently used to automatically construct multidimensional protein phase diagrams to prevent image information loss without complicating the used image classification algorithm. Combining end point and time-dependent features from 3 light sources resulted in a balanced accuracy of 86.4 ± 4.3%, which is comparable to or better than more complex classifiers reported in literature. External validation resulted in a correct formulation classification rate of 91.7%. Subsequent automated construction of the multidimensional protein phase diagrams, using predicted classes, allowed visualization of details such as crystallization rate and protein phase behavior type coexistence.
       
  • Method to Predict Glass Vial Fogging in Lyophilized Drug Products
    • Abstract: Publication date: January 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 1Author(s): Carolin Langer, Hanns-Christian Mahler, Atanas Koulov, Nicolas Marti, Cristina Grigore, Anja Matter, Pascal Chalus, Satish Singh, Thomas Lemazurier, Susanne Joerg, Roman MathaesAbstractGlass fogging is a phenomenon occurring in lyophilized drug products and can be described as a thin product layer deposited on the inner surface of the glass container, in the area not covered by the lyo cake itself. It is often considered a cosmetic defect; however, the loss of container closure integrity is a potential consequence of the fogging’s expansion to the vial neck region, making this a potential critical defect. Thus, a method for predicting the extent of vial fogging before the actual freeze-drying is of particular interest for the pharmaceutical industry. For that reason, we evaluated a simple method (“simulated fogging”) applicable to drug product formulations in a specific container closure system. Two different vial types with different surface hydrophilicity were tested using 3 model protein formulations, comparing the simulated fogging test and the degree of fogging after actual lyophilization. The simulated fogging method could predict fogging and showed a correlation to fogging in lyophilized drug product glass vials. We observed that all formulations showed fogging in the hydrophilic vials. By contrast, hydrophobic vials prevented fogging, however, interestingly with remaining defects of so-called droplet formation. Other than extent of fogging, no additional differences of lyophilized cake properties or other product quality attributes were observed between products using the different glass vial types tested.
       
  • Stability of Protein Formulations at Subzero Temperatures by Isochoric
           Cooling
    • Abstract: Publication date: January 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 1Author(s): Cátia Correia, Evandro Tavares, Carlos Lopes, Joana G. Silva, Andreia Duarte, Vitor Geraldes, Miguel A. Rodrigues, Eduardo P. MeloAbstractOptimization of protein formulations at subzero temperatures is required for many applications such as storage, transport, and lyophilization. Using isochoric cooling (constant volume) is possible to reach subzero temperatures without freezing aqueous solutions. This accelerates protein damage as protein may unfold by cold denaturation and diffusional and conformational freedom is still present. The use of isochoric cooling to faster protein formulations was first demonstrated for the biomedical relevant protein disulfide isomerase A1. Three osmolytes, sucrose, glycerol, and l-arginine, significantly increased the stability of protein disulfide isomerase A1 at -20°C with all tested under isochoric cooling within the short time frame of 700 h. The redox green fluorescent protein 2 was used to evaluate the applicability of isochoric cooling for stability analysis of highly stable proteins. This derivative of GFP is 2.6-fold more stable than the highly stable GFP β-barrel structure. Nevertheless, it was possible to denature a fraction of roGFP2 at −20°C and to assign a stabilizing effect to sucrose. Isochoric cooling was further applied to insulin. Protein damage was evaluated through a signaling event elicited on human hepatocyte carcinoma cells. Insulin at −20°C under isochoric cooling lost 22% of its function after 15 days and 0.6M sucrose prevented insulin deactivation.
       
  • Relation of Colloidal and Conformational Stabilities to Aggregate
           Formation in a Monoclonal Antibody
    • Abstract: Publication date: January 2020Source: Journal of Pharmaceutical Sciences, Volume 109, Issue 1Author(s): Hiroaki Oyama, Hiroki Koga, Takashi Tadokoro, Katsumi Maenaka, Akira Shiota, Masami Yokoyama, Masanori Noda, Tetsuo Torisu, Susumu UchiyamaAbstractAggregation of therapeutic monoclonal antibodies has a potential risk of immunogenicity, requiring minimization of aggregate formation. We have developed a fitting formula for antibody aggregation at 40°C based on physicochemical parameters, including colloidal and conformational stabilities. An IgG1 monoclonal antibody, MAb-T, was formulated in 24 combinations of different buffer types and pH with or without sodium chloride. The fitting formula for monomer loss was successfully established by nonlinear regression analysis of the results from accelerated stability testing. Calculated monomer fraction values by the fitting formula were strongly correlated with experimental values (R2 = 0.92). The model includes secondary virial coefficient, B22, as the representative parameter of colloidal stability, and aggregation temperature, Tagg, representing conformational stability. Then, we examined charge state, conformational flexibility, and thermal unfolding profile of MAb-T to clarify the molecular basis for the different aggregation propensities in sodium acetate buffer and in sodium citrate buffer at the same pH and buffer concentration. We concluded that the accumulation of citrate anions on the surface of MAb-T is the primary source of the less colloidal and conformational stabilities, resulting in the higher aggregation propensity in sodium citrate buffer.
       
  • Comparison of polysorbate 80 hydrolysis and oxidation on the aggregation
           of a monoclonal antibody
    • Abstract: Publication date: Available online 20 November 2019Source: Journal of Pharmaceutical SciencesAuthor(s): Nicholas R. Larson, Yangjie Wei, Indira Prajapati, Aishik Chakraborty, Bjorn Peters, Cavan Kalonia, Suzanne Hudak, Sureshkumar Choudhary, Reza Esfandiary, Prajna Dhar, Christian Schöneich, C. Russell MiddaughAbstractPolysorbates are used ubiquitously in protein therapeutic drugs to help minimize adsorption to surfaces and aggregation. It has been recognized that polysorbate can itself degrade and in turn result in loss of efficacy of therapeutic proteins. We studied the two main pathways of Polysorbate 80 (PS80) degradation, enzymatic ester hydrolysis and oxidation. Degraded polysorbates were quantified through mass spectrometry to identify the loss of individual components. Next Langmuir trough adsorption isotherms were used to characterize changes in the surface activity of the degraded polysorbates. PS80 degraded via hydrolysis results in slower surface adsorption rates, while the oxidized PS80 show increased surface activity. However, the critical micelle concentration remained unchanged. A monoclonal antibody was formulated with stock and degraded polysorbates to probe their ability to prevent aggregation. Hydrolyzed polysorbate resulted in a large increase in particle formation during shaking stress. Oxidized PS80 was still protective against aggregation for the monoclonal antibody. Monomer loss as measured by SEC was comparable in formulations without PS80 to those with esterase hydrolyzed PS80. Monomer loss for oxidized PS80 was similar to that of non-degraded PS80. Hydrolysis of PS80 resulted in free fatty acids which formed insoluble particles during mechanical agitation which stimulated protein aggregation.
       
  • Protein-protein interactions, clustering, and rheology for bovine IgG up
           to high concentrations characterized by small angle x-ray scattering and
           molecular dynamics simulations
    • Abstract: Publication date: Available online 11 November 2019Source: Journal of Pharmaceutical SciencesAuthor(s): Amjad Chowdhury, Geetika Guruprasad, Amy T. Chen, Carl A. Karouta, Marco A. Blanco, Thomas M. Truskett, Keith P. JohnstonAbstractA systematic understanding of intermolecular interactions is necessary for designing concentrated monoclonal and polyclonal antibody solutions with reduced viscosity and enhanced stability. Here, we determine the effects of pH and co-solute on the strength and geometry of short-range anisotropic protein-protein attractions for a polyclonal bovine IgG by comparing intensities (I(q)) obtained from small angle x-ray scattering to those computed in molecular dynamics simulations with 12-bead models. Since our model embodies key features of the protein shape, it can describe the experimental I(q) for solutions of 10-200 mg/mL protein with only a small (
       
  • Cis/trans isomerization of unsaturated fatty acids in polysorbate 80
           during light exposure of a monoclonal antibody-containing formulation
    • Abstract: Publication date: Available online 9 November 2019Source: Journal of Pharmaceutical SciencesAuthor(s): Indira Prajapati, Björn-Hendrik Peters, Nicholas R. Larson, Yangjie Wei, Sureshkumar Choudhary, Cavan Kalonia, Suzanne Hudak, Reza Esfandiary, C. Russell Middaugh, Christian SchöneichAbstractLight exposure of a monoclonal antibody formulation containing polysorbate 80 (PS80) leads to cis/trans isomerization of mono- and polyunsaturated fatty acids. This cis/trans isomerization was monitored by positive electrospray ionization (ESI) mass spectrometry (MS) of intact PS80 components as well as by negative ion ESI MS analysis of free fatty acids (FFAs) generated via esterase-catalyzed hydrolysis. The light-induced cis/trans isomerization of unsaturated fatty acids in PS80 required the presence of the mAb, or, at a minimum (for mechanistic studies), a combination of N-acetyltryptophan amide (NATA) and glutathione disulfide (GSSG), suggesting the involvement of thiyl radicals generated by photo-induced electron transfer from Trp to the disulfide. Product analysis confirmed the conversion of PS80-bound oleic acid to elaidic acid; furthermore, together with linoleic acid we detected conjugated linoleic acids (CLAs) in PS80, which underwent light-induced cis/trans isomerization.
       
  • Algorithm-based liquid formulation development including a DoE concept
           predicts long-term viral vector stability
    • Abstract: Publication date: Available online 2 November 2019Source: Journal of Pharmaceutical SciencesAuthor(s): E.B. Reinauer, S.S. Grosso, S.R. Henz, J.A. Rabas, C. Rodenstein, J. Altrichter, M. Scholz, K. KemterAbstractSpecifically tailored amino acid-based formulations were previously shown to have a high potential to avoid stress-mediated degradation of complex molecules such as monoclonal antibodies and viral vectors. By using adenovirus 5 (Ad5) as a model, we studied whether such formulations may also efficiently protect viral vectors in thermal stress experiments and during long-term liquid storage. Algorithm-based amino acid pre-selection using an excipient database and subsequent application of Design of Experiment (DoE) in combination with a 37 °C challenging model enabled the prediction of long-term storage stability of Ad5. By statistical analysis of the Ad5 infectivity, amino acids with significant influence on Ad5 stability were detected after two and three weeks of liquid storage at 37 °C. Ad5 formulations comprising positively selected amino acids did not reveal any loss of infectivity after 24 months in liquid storage at 5 °C. By contrast, a two log reduction after three months and complete loss of infectivity after 18 months was observed with a standard viral vector formulation. By an optimization round, we designed a simple and well-balanced formulation avoiding MgCl2, previously considered essential in Ad5 formulations. This work demonstrates the efficacy of an algorithm-based development approach in the formulation development for viral vectors.
       
 
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