Rapid, multiplexed detection of the let-7 miRNA family using γPNA amphiphiles in micelle-tagging electrophoresis.

miRNA is a promising class of biomarkers whose levels can be assayed to detect various forms of cancer and other serious diseases. These short, noncoding nucleic acids are difficult to detect due to their low abundance and the marginal stability of their duplexes with DNA probes. In addition, miRNAs within the same family have high sequence homology, and often, related miRNA differ in sequence by only a single base. In this report, we demonstrate an independent detection seven members of the let-7 family of miRNA in a single run. Key to success is the use of mini-PEG-substituted PNA amphiphiles (γPNAA) and highly fluorescent DNA nanotags in micelle tagging electrophoresis (MTE). Multiplexed detection is accomplished in capillary electrophoresis (CE) using oligomeric nanotags of pre-programmed lengths where the presence of a specific miRNA links its nanotag to a micelle drag-tag, which shifts the nanotag elution time to a defined region for detection. We further demonstrate that the peak shape and elution time are unaffected by the presence of up to 10 mg/ml of serum protein in the sample, with a total runtime of less than 4 min and a LOD of 10-100 pM. We discuss efforts to substantially decrease the detection limit using nanotags that are >1000 bp in length.

Representative Scale-Down Lyophilization Cycle Development Using a Seven-Vial Freeze-Dryer (MicroFD®).

We have implemented the use of a small-scale, 7-vial Micro Freeze Dryer (MicroFD®; Millrock Technology, Inc.) that has the capability to accurately control heat transfer during lyophilization. We demonstrate the ability to fine-tune the MicroFD® vial heat transfer coefficient (Kv) to match the Kv of vials in a LyoStar III laboratory-scale unit. When the MicroFD® is run under conditions that match the Kv of the LyoStar III, the resulting lyophilization performance between scales results in equivalent product temperature profiles and critical quality attributes for the same drying process. The proposed workflow demonstrates how exploitation of Kv control in the MicroFD® enables cycle development of at-scale lyophilization processes using only 7 product vials. By changing the MicroFD®Kv, laboratory and, potentially, manufacturing cycles may be simulated using only 7 product vials for tremendous active pharmaceutical ingredient savings, as long as at-scale heat transfer coefficients are well characterized.

Optimization of Primary Drying in Lyophilization During Early-Phase Drug Development Using a Definitive Screening Design With Formulation and Process Factors.

Development of optimal drug product (DP) lyophilization cycles is typically accomplished via multiple engineering runs to determine appropriate process parameters. These runs require significant time and product investments, which are especially costly during early phase development when the DP formulation and lyophilization process are often defined simultaneously. Even small changes in the formulation may require a new set of engineering runs to define lyophilization process parameters. To overcome these development difficulties, an 8 factor definitive screening design, including both formulation and process parameters, was executed on a fully human monoclonal antibody DP. The definitive screening design enables evaluation of several interdependent factors to define critical parameters that affect primary drying time and product temperature. From these parameters, a lyophilization development model is defined where near optimal process parameters can be derived for many different DP formulations. This concept is demonstrated on a monoclonal antibody DP where statistically predicted cycle responses agree well with those measured experimentally. This design of experiments approach for early phase lyophilization cycle development offers a workflow that significantly decreases the development time of clinically and potentially commercially viable lyophilization cycles for a platform formulation that still has variable range of compositions.

High affinity γPNA sandwich hybridization assay for rapid detection of short nucleic acid targets with single mismatch discrimination.

Hybridization analysis of short DNA and RNA targets presents many challenges for detection. The commonly employed sandwich hybridization approach cannot be implemented for these short targets due to insufficient probe-target binding strengths for unmodified DNA probes. Here, we present a method capable of rapid and stable sandwich hybridization detection for 22 nucleotide DNA and RNA targets. Stable hybridization is achieved using an n-alkylated, polyethylene glycol γ-carbon modified peptide nucleic acid (γPNA) amphiphile. The γPNA's exceptionally high affinity enables stable hybridization of a second DNA-based probe to the remaining bases of the short target. Upon hybridization of both probes, an electrophoretic mobility shift is measured via interaction of the n-alkane modification on the γPNA with capillary electrophoresis running buffer containing nonionic surfactant micelles. We find that sandwich hybridization of both probes is stable under multiple binding configurations and demonstrate single base mismatch discrimination. The binding strength of both probes is also stabilized via coaxial stacking on adjacent hybridization to targets. We conclude with a discussion on the implementation of the proposed sandwich hybridization assay as a high-throughput microRNA detection method.