Multi-dose Formulation Development for a Quadrivalent Human Papillomavirus Virus-Like Particle-Based Vaccine: Part II- Real-time and Accelerated Stability Studies.

This work describes Part 2 of multi-dose formulation development of a Human Papillomavirus (HPV) Virus-Like Particle (VLP) based vaccine (see Part 1 in companion paper). Storage stability studies with candidate multi-dose formulations containing individual or combinations of seven different antimicrobial preservatives (APs) were performed with quadrivalent HPV VLP (6, 11, 16, 18) antigens adsorbed to aluminum-salt adjuvant (Alhydrogel®). Real-time (up to two years, 2-8°C) and accelerated (months at 25 and 40°C) stability studies identified eight lead candidates as measured by antigen stability (competitive ELISA employing conformational serotype-specific mAbs), antimicrobial effectiveness (modified European Pharmacopeia assay), total protein content (SDS-PAGE), and AP concentration (RP-UHPLC).

Analytical and Preformulation Characterization Studies of Human Papillomavirus Virus-Like Particles to Enable Quadrivalent Multi-Dose Vaccine Formulation Development.

Introducing multi-dose formulations of Human Papillomavirus (HPV) vaccines will reduce costs and enable improved global vaccine coverage, especially in low- and middle-income countries. This work describes the development of key analytical methods later utilized for HPV vaccine multi-dose formulation development. First, down-selection of physicochemical methods suitable for multi-dose formulation development of four HPV (6, 11, 16, and 18) Virus-Like Particles (VLPs) adsorbed to an aluminum adjuvant (Alhydrogel®, AH) was performed.

Modeling the long-term 2-8 °C stability profiles of a live, rotavirus vaccine candidate (RV3-BB) in various liquid formulations via extrapolations of real-time and accelerated stability data.

To accelerate the formulation development of live-virus vaccine (LVV) candidates, more rapid approaches to rank-order formulations and estimate their real-time storage stability losses are needed. In this case-study, we utilize new and previously described stability data of a live, rotavirus vaccine candidate (RV3-BB) in three different liquid formulations to model and compare predicted vs. experimental RV3-BB stability profiles.

Formulation Development and Improved Stability of a Combination Measles and Rubella Live-Viral Vaccine Dried for Use in the Nanopatch Microneedle Delivery System.

Measles (Me) and rubella (Ru) viral diseases are targeted for elimination by ensuring a high level of vaccination coverage worldwide. Less costly, more convenient MeRu vaccine delivery systems should improve global vaccine coverage, especially in low - and middle - income countries (LMICs). In this work, we examine formulating a live, attenuated Me and Ru combination viral vaccine with Nanopatch™, a solid polymer micro-projection array for intradermal delivery.

Formulation development of a live attenuated human rotavirus (RV3-BB) vaccine candidate for use in low- and middle-income countries.

Formulation development was performed with the live, attenuated, human neonatal rotavirus vaccine candidate (RV3-BB) with three main objectives to facilitate use in low- and middle- income countries including (1) a liquid, 2-8°C stable vaccine, (2) no necessity for pre-neutralization of gastric acid prior to oral administration of a small-volume dose, and (3) a low-cost vaccine dosage form. Implementation of a high-throughput RT-qPCR viral infectivity assay for RV3-BB, which correlated well with traditional FFA assays in terms of monitoring RV3-BB stability profiles, enabled more rapid and comprehensive formulation development studies. A wide variety of different classes and types of pharmaceutical excipients were screened for their ability to stabilize RV3-BB during exposure to elevated temperatures, freeze-thaw and agitation stresses.

Developing a manufacturing process to deliver a cost effective and stable liquid human rotavirus vaccine.

Despite solid evidence of the success of rotavirus vaccines in saving children from fatal gastroenteritis, more than 82 million infants worldwide still lack access to a rotavirus vaccine. The main barriers to global rotavirus vaccine coverage include cost, manufacturing capacity and suboptimal efficacy in low- and lower-middle income countries. One vaccine candidate with the potential to address the latter is based on the novel, naturally attenuated RV3 strain of rotavirus, RV3-BB vaccine administered in a birth dose strategy had a vaccine efficacy against severe rotavirus gastroenteritis of 94% at 12 months of age in infants in Indonesia.

Effect of Formulation Variables on the Stability of a Live, Rotavirus (RV3-BB) Vaccine Candidate using in vitro Gastric Digestion Models to Mimic Oral Delivery.

In this work, two different in vitro gastric digestion models were used to evaluate the stability of a live attenuated rotavirus vaccine candidate (RV3-BB) under conditions designed to mimic oral delivery in infants. First, a forced-degradation model was established at low pH to assess the buffering capacity of formulation excipients and to screen for RV3-BB stabilizers. Second, a sequential-addition model was implemented to examine RV3-BB stability under conditions more representative of oral administration to infants.

Discrete microfluidics for the isolation of circulating tumor cell subpopulations targeting fibroblast activation protein alpha and epithelial cell adhesion molecule.

Circulating tumor cells consist of phenotypically distinct subpopulations that originate from the tumor microenvironment. We report a circulating tumor cell dual selection assay that uses discrete microfluidics to select circulating tumor cell subpopulations from a single blood sample; circulating tumor cells expressing the established marker epithelial cell adhesion molecule and a new marker, fibroblast activation protein alpha, were evaluated. Both circulating tumor cell subpopulations were detected in metastatic ovarian, colorectal, prostate, breast, and pancreatic cancer patients and 90% of the isolated circulating tumor cells did not co-express both antigens.

Characterization of activated cyclic olefin copolymer: effects of ethylene/norbornene content on the physiochemical properties.

The ethylene/norbornene content within cyclic olefin copolymer (COC) is well known to affect the chemical and physical properties of the copolymer, such as the glass transition temperature (T) and transparency. However, no work has been reported evaluating the effects of the ethylene/norbornene content on the surface properties of COC following UV/O or O plasma activation. Activation with either O plasma or UV/O is often used to assist in thermal assembly of fluidic devices, increasing the wettability of the surfaces, or generating functional scaffolds for the attachment of biological elements.

Current and future bioanalytical approaches for stroke assessment.

Efforts are underway to develop novel platforms for stroke diagnosis to meet the criteria for effective treatment within the narrow time window mandated by the FDA-approved therapeutic (<3 h). Blood-based biomarkers could be used for rapid stroke diagnosis and coupled with new analytical tools, could serve as an attractive platform for managing stroke-related diseases. In this review, we will discuss the physiological processes associated with stroke and current diagnostic tools as well as their associated shortcomings.

Recent and near-future advances in nucleic acid-based diagnosis of stroke.

Stroke is a leading cause of death and disability in adults, but at present, treatment for ischemic stroke reaches only a small percentage of patients. This is because of the very short time window for treatment and the time-consuming evaluation involved. Intense efforts are underway to find novel approaches to expedite stroke diagnosis and treatment.

Surface charge, electroosmotic flow and DNA extension in chemically modified thermoplastic nanoslits and nanochannels.

Thermoplastics have become attractive alternatives to glass/quartz for microfluidics, but the realization of thermoplastic nanofluidic devices has been slow in spite of the rather simple fabrication techniques that can be used to produce these devices. This slow transition has in part been attributed to insufficient understanding of surface charge effects on the transport properties of single molecules through thermoplastic nanochannels. We report the surface modification of thermoplastic nanochannels and an assessment of the associated surface charge density, zeta potential and electroosmotic flow (EOF).

Parallel affinity-based isolation of leukocyte subsets using microfluidics: application for stroke diagnosis.

We report the design and performance of a polymer microfluidic device that can affinity select multiple types of biological cells simultaneously with sufficient recovery and purity to allow for the expression profiling of mRNA isolated from these cells. The microfluidic device consisted of four independent selection beds with curvilinear channels that were 25 μm wide and 80 μm deep and were modified with antibodies targeting antigens specifically expressed by two different cell types. Bifurcated and Z-configured device geometries were evaluated for cell selection.

UV activation of polymeric high aspect ratio microstructures: ramifications in antibody surface loading for circulating tumor cell selection.

The need to activate thermoplastic surfaces using robust and efficient methods has been driven by the fact that replication techniques can be used to produce microfluidic devices in a high production mode and at low cost, making polymer microfluidics invaluable for in vitro diagnostics, such as circulating tumor cell (CTC) analysis, where device disposability is critical to mitigate artifacts associated with sample carryover. Modifying the surface chemistry of thermoplastic devices through activation techniques can be used to increase the wettability of the surface or to produce functional scaffolds to allow for the covalent attachment of biologics, such as antibodies for CTC recognition. Extensive surface characterization tools were used to investigate UV activation of various surfaces to produce uniform and high surface coverage of functional groups, such as carboxylic acids in microchannels of different aspect ratios.

Simple replication methods for producing nanoslits in thermoplastics and the transport dynamics of double-stranded DNA through these slits.

Mixed-scale nano- and microfluidic networks were fabricated in thermoplastics using simple and robust methods that did not require the use of sophisticated equipment to produce the nanostructures. High-precision micromilling (HPMM) and photolithography were used to generate mixed-scale molding tools that were subsequently used for producing fluidic networks into thermoplastics such as poly(methyl methacrylate), PMMA, cyclic olefin copolymer, COC, and polycarbonate, PC. Nanoslit arrays were imprinted into the polymer using a nanoimprinting tool, which was composed of an optical mask with patterns that were 2-7 µm in width and a depth defined by the Cr layer (100 nm), which was deposited onto glass.