Formulation Screening Research Articles

Use of high throughput screening methods to study dual-functional crosslinkable latexes

Structure-formulation-property relationships for crosslinkable core-shell latexes were studied initially using high throughput screening methods and then using conventional experimental methods to determine optimum coating formulations. Two approaches that are used within coatings technology to improve sustainability are reduction in volatile organic compounds (VOC) and reduction in energy needed to cure coatings. Hybrid crosslinkable core-shell latexes have shown potential in meeting these challenges where film properties provided by particle coalescence are not sufficient and chemical crosslinking is needed. However, with numerous parameters involved such as latex particle architecture, nature and amount of crosslinker, and curing conditions, the screening of formulations via conventional coating testing methods becomes laborious and time-consuming. The results from high-throughput dye extraction and puncture testing were compared to data from conventional testing methods such as chemical resistance (MEK double rubs), pendulum hardness, and tensile testing to identify correlations. The dye extraction results have shown a strong correlation to solvent resistance and pendulum hardness data whereas toughness from puncture testing data correlated to tensile testing. Both high throughput dye extraction and puncture testing methods can be used for the accelerated screening of crosslinkable coating formulations to optimize curing parameters to achieve the best coating performance.

The importance of spheroids in analyzing nanomedicine efficacy.

The use of nanomedicines for cancer treatment holds a great potential due to their improved efficacy and safety. During the nanomedicine preclinical in vitro evaluation stage, these are mainly tested on cell culture monolayers. However, these 2D models are an unrealistic representation of the in vivo tumors, leading to an inaccurate screening of the candidate formulations. To address this problem, spheroids are emerging as an additional tool to validate the efficacy of new therapeutics due to the ability of these 3D in vitro cancer models to mimic the key features displayed by in vivo solid tumors. In this review, the application of spheroids for the evaluation of nanomedicines' physicochemical properties and therapeutic efficacy is discussed.

Formulation, Characterization and Biological Activity Screening of Sodium Alginate-Gum Arabic Nanoparticles Loaded with Curcumin.

The approach of drug delivery systems emphasizes the use of nanoparticles as a vehicle, offering the optional property of delivering drugs as a single dose rather than in multiple doses. The current study aims to improve antioxidant and drug release properties of curcumin loaded gum Arabic-sodium alginate nanoparticles (Cur/ALG-GANPs). The Cur/ALG-GANPs were prepared using the ionotropic gelation technique and further subjected to physico-chemical characterization using attenuated total reflectance–Fourier transform infrared (ATR-FTIR), X-ray diffractometry (XRD), differential scanning calorimetry (DSC), size distribution, and transmission electron microscopy (TEM). The size of Cur/ALG-GANPs ranged between 10 ± 0.3 nm and 190 ± 0.1 nm and the zeta potential was –15 ± 0.2 mV. The antioxidant study of Cur/ALG-GANPs exhibited effective radical scavenging capacity for 1,1-diphenyl-2-picrylhydrazyl (DPPH) at concentrations that ranged between 30 and 500µg/mL. Cytotoxicity was performed using MTT assay to measure their potential in inhibiting the cell growth and the result demonstrated a significant anticancer activity of Cur/ALG-GANPs against human liver cancer cells (HepG2) than in colon cancer (HT29), lung cancer (A549) and breast cancer (MCF7) cells. Thus, this study indicates that Cur/ALG-GANPs have promising anticancer properties that might aid in future cancer therapy.

Robotically handled whole-tissue culture system for the screening of oral drug formulations.

Monolayers of cancer-derived cell lines are widely used in the modelling of the gastrointestinal (GI) absorption of drugs and in oral drug development. However, they do not generally predict drug absorption in vivo. Here, we report a robotically handled system that uses large porcine GI tissue explants that are functionally maintained for an extended period in culture for the high-throughput interrogation (several thousand samples per day) of whole segments of the GI tract. The automated culture system provided higher predictability of drug absorption in the human GI tract than a Caco-2 Transwell system (Spearman's correlation coefficients of 0.906 and 0.302, respectively). By using the culture system to analyse the intestinal absorption of 2,930 formulations of the peptide drug oxytocin, we discovered an absorption enhancer that resulted in a 11.3-fold increase in the oral bioavailability of oxytocin in pigs in the absence of cellular disruption of the intestinal tissue. The robotically handled whole-tissue culture system should help advance the development of oral drug formulations and might also be useful for drug screening applications.

On‐Chip Electrical Monitoring of Real‐Time “Soft” and “Hard” Protein Corona Formation on Carbon Nanoparticles

AbstractWhen nanoparticles encounter a biological fluid, proteins, lipids, and other biomolecules adsorb on the nanoscale surface consequently leading to the evolution of a protein shell or “corona.” The corona formed is dynamic in nature and depends on the “synthetic identity” of the NPs, ultimately affecting their biological response. In this paper, an integrated microfluidic platform coupled with an electrical resistance measurement setup is developed to monitor and investigate the real‐time formation of a biomolecular corona of carbon nanoparticles. “Soft” and “hard” corona formation stages are effectively discriminated based on their nanoscale surface chemistries when combined with a time‐frequency tool known as wavelet transform and machine‐learning techniques. Additionally, the corona and its composition are studied using different techniques such as dynamic light scattering, nanoparticle tracking analysis, zeta potential, excitation–emission profiles, 1D sodium dodecyl polyacrylamide gel electrophoresis and subsequently, liquid chromatography‐mass spectrometry analysis. The dynamic setup can eventually be used as a valuable tool for screening of any nanoparticles formulations with distinct surface chemistries for the purpose of reduced protein adsorption/weaker protein corona formation and consequently enhance the success of targeted drug delivery.

Surfactant-polymer flooding formulated with commercial surfactants and enhanced by negative salinity gradient

In this study, a high-performance surfactant-polymer (SP) formulation was developed using commercially available surfactants instead of lab-synthesized ones with limited sources. Alcohol propoxysulfates (AES-1 and AES-2), cocamidopropyl hydroxysultaine (CAS), alkyl benzene sulfonate (ABS), and alpha olefin sulfonate (AOS) were mixed in pairs for the formulation screening. The AES-1/ABS formulation with a mixing ratio of 6/4 was able to achieve ultralow interfacial tension (IFT) and possessed excellent aqueous stability at additional NaCl concentration greater than 1.25 wt%. Phase behavior tests illustrated that the optimum salinity (1.6 wt%) and salinity range (1.5 ~ 1.8 wt%) for type III microemulsions were nearly unchanged in spite of the water-oil ratio variation as no in situ soap was generated in the absence of alkali. To investigate the effect of different salinity profiles on the post-polymer SP flooding, 3 corefloods were conducted at 0.3 wt% surfactant concentration. Highest incremental oil recovery (34.8% OOIP) by SP flooding was obtained in coreflood No.2 in which 1 PV NaCl preflush, 0.3 PV NaCl-SP slug, and 0.2 PV polymer drive with additional NaCl of 2.0 wt%, 1.6 wt% and 0.5 wt% were successively injected. Reducing preflush volume in coreflood No.1 and No.3 resulted in lower incremental oil recovery. Effluent TDS analysis indicated that only in coreflood No.2 the maximum effluent salinity (22,249 ppm) exceeded optimum salinity (about 21,000 ppm) and negative salinity gradient concept was realized in the whole SP flooding process. With sufficient salinity preflush, the AES-1/ABS formulation achieved a cumulative oil recovery as high as 93.9% OOIP after polymer flooding.

Rapid Screening of Additive Formulations for Enhancing Xylanase Stability in Pulp Bleaching and Storage Conditions

Enzyme deactivation is a significant parameter to restrain the industrial commercialization of enzymes. In this study, a high-throughput screening technique using differential scanning fluorimetry (DSF) incorporated with enzyme activity analysis was thus developed in order to identify a suitable additive formulation for promoting enzyme stability in terms of conformational stability and functional activity. To demonstrate the concept feasibility, endo-xylanase obtained from metagenome of termite gut-inhabiting bacteria (XYN12) was used as a study model and several group of additives including salts, polymers, polyols and sugars at various concentrations and different combination patterns (single, double and triple pairing) were applied for investigating their effects on enzyme stability. As a result, a total of 80 reaction mixtures were retrieved including a native control (enzyme without any additives). DSF assay was performed using real-time PCR instrument to detect unfolding of protein in the presence of a florescence dye. Accurate melting temperature (Tm) values of each enzyme formulation were calculated using Boltzmann equation and compared to the Tm of the native control in order to find ?Tm. After that, the residual activity of xylanase in all reaction mixtures were measured using pHBAH method in automated liquid handling system. For single additives, glycerol had a stabilizing effect to the target enzyme, as observed by a significant increase in the ?Tm, while ?(NH4)2SO4, CaCl2, NaCl destabilized it. In addition, cocktail additives included glycerol, PEG4000 and trehalose dramatically enhanced the enzyme stability by positive shifting of Tm from 54 to 56 (C and increasing of relative activity by more than 4 folds compared to single additive. Storage stability of the enzyme was improved when mixed with this cocktail additive as indicated by remaining of the enzyme activity (86 %) after kept at 30 (C, pH 7.4 for 262 days, whereas native control lost almost of its activity within 40 days when stored at the same condition.

Understanding the freezing responses of T cells and other subsets of human peripheral blood mononuclear cells using DSMO-free cryoprotectants

BackgroundThis study examined the freezing responses of peripheral blood mononuclear cells (PBMCs) and specific white blood cell subsets contained therein when cryopreserved in three combinations of osmolytes composed of sugars, sugar alcohols and amino acids. MethodsA differential evolution algorithm with multiple objectives was used to optimize cryoprotectant composition and thus the post-thaw recoveries for both helper and cytotoxicity T cells simultaneously. ResultsThe screening of various formulations using a differential evolution algorithm showed post-thaw recoveries greater than 80% for the two subsets of T cells. The phenotypes and viabilities of PBMC subsets were characterized using flow cytometry. Significant differences between the post-thaw recovery for helper T cells and cytotoxic T cells were observed. Statistical models were used to analyze the importance of individual osmolytes and interactions between post-thaw recoveries of three subsets of T cell including helper T cells, cytotoxic T cells and natural killer T cells. The statistical model indicated that the preferred concentration levels of osmolytes and interaction modes were distinct between the three subsets studied. PBMCs were cultured for 72 h post-thaw to determine the stability of the cells. Because post-thaw apoptosis is a significant concern for lymphocytes, apoptosis of helper T cell and cytotoxic T cells frozen in a DMSO-free cryoprotectant was analyzed immediately post-thaw and 24 h post-thaw. Both cell types showed a decrease in cell viability 24 h post-thaw compared with immediately post-thaw. Helper T cell viability dropped 17%, and cytotoxic T cells had a 10% drop in viability. Immediately post-thaw, both cell types had >30% of cells in early apoptosis, but after 24 h the number of cells in early apoptosis decreased to below 20%. ConclusionThis study helped us identify the freezing responses of different human PBMC subsets using combinations of osmolytes.

Improved encapsulation efficiency and storage stability of spray dried microencapsulated lutein with carbohydrates combinations as encapsulating material

Lutein is utilized in food industry as a natural colorant or functional ingredient. Nevertheless, its application is limited by its chemical instability. The aim of this research was to develop and optimize lutein microencapsulated powders (LMPs) by spray drying to enhance storage stability of lutein. In formulation screening, LMPs, with inulin and modified starch as combined encapsulating materials (EMs), exhibited maximum encapsulation efficiency (EE) of 80.0 ± 0.6% and retention value (RV) of 87.9 ± 0.4%. The preparation process was further optimized by Box-Behnken design with EMs concentration, proportion of inulin and modified starch as EMs and inlet-drying temperature as crucial factors. EE, RV and product yield (PY) as critical quality attributes were considered. The micro-structural characterizations of six experimental LMPs were observed by scanning electron microscopy and used to describe the effects of the three crucial factors on morphological characterization of LMPs. Subsequently, the optimal condition for preparing LMPs was identified by response surface analysis. The actual EE, RV and PY values of the optimal LMPs were measured to be 85.0 ± 0.5, 90.1 ± 0.4 and 91.2 ± 0.7%, respectively. In the stability assessment, temperature degradation kinetics of microencapsulated lutein was analyzed. The chemical stability of the optimal LMPs significantly increased after exposure to four different temperatures.

Toxicity and gas chromatography-mass spectrometry analyses of a polyherbal formulation commonly used in Ibadan metropolis, Nigeria

A polyherbal formulation mainly composed of Hibiscus sabdariffa and Aloe barbadensis commonly patronized by some staff and students of a College Hospital in Ibadan, Nigeria was evaluated for its toxicity status and bioactive constituents. Its safety was assessed using acute and sub-chronic toxicity models in Wistar rats while Gas Chromatography-Mass Spectrometry (GC–MS) was used to identify the bioactive constituents. Findings showed that oral administration of the polyherbal formulation did not cause any notable behavioral changes or mortality in the animals during the acute toxicity testing. Similarly, statistical analysis of the data obtained showed that sub-chronic administration of the polyherbal formulation did not cause any significant changes in the serum activities of liver-function enzymes, hematological markers, serum electrolytes and other evaluated blood-chemistry indices in the experimental animals compared to those of their control counterparts. These observations were substantiated by the liver photomicrographs which showed that treatment of animals with the polyherbal mixture did not induce any visible lesions or derangements in the examined organ. Besides, GC–MS screening of the polyherbal formulation revealed compounds with known biological effects which are associable with the locally acclaimed therapeutic applications of the formulation. The outcome of this study therefore suggests high safety for the investigated polyherbal mixture and thus supports its usage in folklore medicine.

Overview of Safety Assessment and Toxicological Screening of Dermal Formulations

Overview of Safety Assessment and Toxicological Screening of Dermal Formulations

Challenges and Considerations in Development and Manufacturing of High Concentration Biologics Drug Products

With the advent of novel biotherapeutics in indications such as oncology, the attention has moved to making these therapies patient centric, potentially available for self-administration. Given the volume limitations of the other convenient routes of administration such a subcutaneous, intramuscular, there is a need for high concentration drug products. Along with the concerns of inherent protein instability at high concentration, there are other practical challenges during the development of these drug products. This review focuses on the considerations in formulation screening, process development, manufacturing, analytical methods and stability for high concentration liquid drug product. For the scenarios where, high concentration liquid drug product is not feasible, alternate approaches can be undertaken to obtain high concentration drug product during administration. The discussion on the current available alternate technologies with an overview of their advantages and disadvantages are covered here. The scope of the overall discussions is introductory and limited to cautionary notes and experiences as opposed to prescriptive recommendations and detailed literature reviews: our primary goal is to provide a compiled set of considerations that can guide a detailed literature research and a to-do list for high concentration development and manufacturing activities. This manuscript aims to highlight the motivations to develop high concentration parenteral protein drug products, a wide range of -commonly known or sometimes overlooked- challenges associated with their development and manufacturing, as well as some approaches towards mitigation of said challenges.

Automated and Continuous Production of Polymeric Nanoparticles.

Polymeric nanoparticles (NPs) are increasingly used as therapeutics, diagnostics, and building blocks in (bio)materials science. Current barriers to translation are limited control over NP physicochemical properties and robust scale-up of their production. Flow-based devices have emerged for controlled production of polymeric NPs, both for rapid formulation screening (~μg min−1) and on-scale production (~mg min−1). While flow-based devices have improved NP production compared to traditional batch processes, automated processes are desired for robust NP production at scale. Therefore, we engineered an automated coaxial jet mixer (CJM), which controlled the mixing of an organic stream containing block copolymer and an aqueous stream, for the continuous nanoprecipitation of polymeric NPs. The CJM was operated stably under computer control for up to 24 h and automated control over the flow conditions tuned poly(ethylene glycol)-block-polylactide (PEG5K-b-PLA20K) NP size between ≈56 nm and ≈79 nm. In addition, the automated CJM enabled production of NPs of similar size (Dh ≈ 50 nm) from chemically diverse block copolymers, PEG5K-b-PLA20K, PEG-block-poly(lactide-co-glycolide) (PEG5K-b-PLGA20K), and PEG-block-polycaprolactone (PEG5K-b-PCL20K), by tuning the flow conditions for each block copolymer. Further, the automated CJM was used to produce model nanotherapeutics in a reproducible manner without user intervention. Finally, NPs produced with the automated CJM were used to scale the formation of injectable polymer–nanoparticle (PNP) hydrogels, without modifying the mechanical properties of the PNP gel. In conclusion, the automated CJM enabled stable, tunable, and continuous production of polymeric NPs, which are needed for the scale-up and translation of this important class of biomaterials.

Insecticidal Gene Silencing by RNAi in the Neotropical Region.

Insecticidal gene silencing by RNA interference (RNAi) involves a post-transcriptional mechanism with great potential for insect control. Here, we aim to summarize the progress on RNAi research toward control of insect pests in the Neotropical region and discuss factors determining its efficacy and prospects for pest management. We include an overview of the available RNAi information for Neotropical pests in the Lepidoptera, Coleoptera, Diptera, and Hemiptera orders. Emphasis is put on significant findings in the use of RNAi against relevant Neotropical pests, including diamondback moth (Plutella xylostella L.), Asian citrus psyllid (Diaphorina citri Kuwayama), and the cotton boll weevil (Anthonomus grandis Boheman). We also examine the main factors involved in insecticidal RNAi efficiency and major advances to improve screening of lethal genes, formulation, and delivery. Few studies detail resistance mechanisms to RNAi, demonstrating a need for more research. Advances in formulation, delivery, and resistance management tools for insecticidal RNAi in the Neotropics can provide a basis for efficient field application.

Flow‐based reactor design for the continuous production of polymeric nanoparticles

AbstractPolymeric nanoparticles (NPs) are versatile and effective drug delivery systems (DDS) that can be produced via nanoprecipitation of block copolymers. Yet, translation into clinical products has been limited. Thus, methods for NP production that enable rapid formulation screening and continuous production are needed. Toward this end, we engineered a coaxial jet mixer (CJM) for controlled and continuous nanoprecipitation in flow. The CJM enabled continuous assembly of poly(ethylene glycol)‐block‐polylactide NPs with various co‐solvents and was compared to batch nanoprecipitation. Other fabricated microfluidic devices were suitable for small scale formulation screening but more limited in scalable and continuous processes. In contrast, the CJM was tolerant to all water‐miscible solvents tested, enabled formulation screening, and scalable production of NPs and DDS. In total, the CJM provides a complementary approach to the process engineering of polymeric NP formation that can be used broadly for formulation screening and production.

Using LC-MS to Identify Clipping in Self-Assembled Nanoparticles During Vaccine Development.

A hemagglutinin stabilized stem nanoparticle (HA-SS-np) that is designed to provide broad protection against influenza is being developed as a potential vaccine. During an early formulation screening study, reducing gel (rCGE) analysis indicated product degradation in a few candidate buffers at the first-week accelerated stability point, whereas no change was shown in the size exclusion chromatography (SEC) measurement. A LC-MS workflow was therefore applied to investigate the integrity of this large HA-SS-np vaccine molecule (≈ 1MDa). Application of LC-MS was critical to rationalize the conflicting results from the rCGE and SEC assays and led to the discovery that (1) an unexpected sequence clipping in the HA-SS-np subunits occurred, explaining the atypical reducing gel profile, and (2) an undisrupted disulfide bond held the two fragments together, explaining the unchanged SEC profile. This analytical case study led to a formulation buffer redesign, which mitigated the issue.

Whole-Eye Perfusion Model for Screening of the Ocular Formulations via Confocal Laser Scanning Microscopy.

Various physiological, anatomical barriers make ocular drug delivery very challenging. Hence, better in vitro screening models are needed for rapid screening of the formulations. In this study, a simple whole-eye perfusion model was designed and its application was explored for screening targeted formulation across the full-thickness cornea using confocal laser scanning microscopy. PEG-cholecalciferol-based integrin targeted coumarin-6 micelles (TC6M) and non-targeted coumarin-6 micelles (NTC6M) were developed by solvent diffusion evaporation technique. The formulations NTC6M and TC6M had particles size 23.5 ± 5nm and 28.5 ± 6nm respectively and osmolality of 294-300mOsml/Kg. The whole-eye perfusion model was developed using porcine eye. TC6M and NTC6M were instilled on the excised porcine eyes as well as in the eyes of NZW rabbits. Corneas were excised from the experimental eyes; coumarin-6 penetration across the corneas was analyzed using confocal microscope. Coumarin-6-loaded micelles had particle size below 50nm. NTC6M formulations showed penetration to the deeper layers up to 500μm porcine eyes and up to 50μm in rabbit corneas. However, TC6M formulations exhibited superior retention, as higher fluorescent intensities were observed in upper layers up to 50μm depth in the porcine eye and 20μm depth in rabbit eye. Hence, applicability of whole-eye perfusion model in preliminary screening of the formulations was successfully demonstrated. Whole-eye perfusion model when combined with confocal microscopy has potential to be used as an efficient tool for rapid screening and optimization of various ophthalmic formulations.

Photochemistry with Cyanines in the Near Infrared: A Step to Chemistry 4.0 Technologies.

Cyanines covering the absorption in the near infrared (NIR) are attractive for distinct applications. They can interact either with lasers exhibiting line‐shaped focus emitting at both 808 and 980 nm or bright high intensity NIR‐LEDs with 805 nm emission, respectively. This is drawing attention to Industry 4.0 applications. The major deactivation occurs through a non‐radiative process resulting in the release of heat into the surrounding, although a small fraction of radiative deactivation also takes place. Most of these NIR‐sensitive systems possess an internal activation barrier to react in a photonic process with initiators resulting in the generation of reactive radicals and acidic cations. Thus, the heat released by the NIR absorber helps to bring the system, consisting of an NIR sensitizer and initiator, above such internal barriers. Molecular design strategies making these systems more compatible with distinct applications in a certain oleophilic surrounding are considered as a big challenge. This includes variations of the molecular pattern and counter ions derived from super acids exhibiting low coordinating properties. Further discussion focusses on the use of such systems in Chemistry 4.0 related applications. Intelligent software tools help to improve and optimize these systems combining chemistry, engineering based on high‐throughput formulation screening (HTFS) technologies, and machine learning algorithms to open up novel solutions in material sciences.

A LC–MS/MS platform for the identification of productivity markers in industrial mammalian cell culture media

Abstract Cell culture media formulations supplemented with soy hydrolysates for commercial scale manufacturing of therapeutic proteins were characterized by high-resolution LC–MS/MS to identify specific media components that contributed to cellular bioprocess variability. Initial untargeted screening of media formulations resulted in the combined quantification of >2500 features upon analysis by reversed-phase and hydrophilic interaction liquid chromatography. Chemometric evaluation of quantified features by Principal Component Analysis and Hierarchical Clustering revealed clear correlation between media lots and the corresponding hydrolysates implemented during formulation development stages, suggesting that the observed variation in culture performance could be influenced by components present within hydrolysates. Through multivariate data analysis, a small number of dipeptides, identified as Tyrosyl-Leucine, Phenylalanyl-Valine and LL-Cyclo(Leucylprolyl) were determined in media as potential contributors to bioprocess variability due to their statistically significant higher abundance in formulations which yielded low cellular productivity. Subsequent quantification of these features was performed through targeted LC-QQQ-MS/MS which however, revealed discrepancies between dipeptide concentration levels in media and corresponding hydrolysates presumably due to their potential instability upon interaction with other chemically defined components such as metals or chelating agents. The nutritional impact of the aforementioned features of interest on CHO cell growth performance was assessed through spiking studies in representative shake-flask cultures which however, failed to demonstrate clear correlation between dipeptide concentrations and achieved product yields further indicating the presence of a plausible synergetic effect between the identified dipeptides and alternative media components.

Redesigning food protein formulations with empirical phase diagrams: A case study on glycerol-poor and glycerol-free formulations

Redesigning existing food protein formulations is necessary in situations where food authorities propose dose adjustments or removal of currently employed additives. Redesigning formulations involves evaluating substitute additives to obtain similar long-term physical stability as the original formulation. Such formulation screening experiments benefit from comprehensive data visualization, understanding the effects of substitute additives on long-term physical stability, and identification of short-term optimization targets. This work employs empirical phase diagrams to reach these benefits by combining multidimensional long-term protein physical stability data with short-term empirical protein properties. A case study was performed where multidimensional protein phase diagrams (1152 formulations) allowed for identification of stabilizing effects as a result of pH, methionine, sugars, salt, and minimized glycerol content. Corresponding empirical protein property diagrams (144 formulations) resulted in the identification of normalized surface tension as a short-term empirical protein property to reach long-term physical stability presumably similar to the original product, namely via preferential hydration. Additionally, changes in pH and salt were identified as environmental optimization targets to reach stability via repulsive electrostatic forces. This case study shows the applicability of the empirical phase diagram method to rationally perform formulation redesign screenings, while simultaneously expanding knowledge on protein long-term physical stability.