Synthetic biology: advancing biological frontiers by building synthetic systems

Advances in synthetic biology are contributing to diverse research areas, from basic biology to biomanufacturing and disease therapy. We discuss the theoretical foundation, applications, and potential of this emerging field

UV-Photolithography Fabrication of Poly-Ethylene Glycol Hydrogels Encapsulated with Hepatocytes

The development of biomanufacturing technologies particularly, layered manufacturing has advanced cell encapsulation processes in an effort to mimic the cellular microenvironment for invitro studies. This paper illustrates an inexpensive UV-photolithographic method for encapsulation of human hepatocytes in three dimensional structures using poly-ethylene diacrylate (PEGDA) hydrogels as candidate substrates. In order to further develop this technology for layered fabrication, we have quantified the long-term effects of the photo-initiator concentration and UV light exposure on the metabolic rates of encapsulated human hepatocytes under a 21 day study. The photoinitator toxicity was observed immediately after polymerization with no significant cytotoxicity on a long term basis. A cellular viability is examined and reported for the UV photopolymerization process. Cell phenotype maintenance was observed by measuring the amount of urea produced over a 1 week time period. This photo encapsulation process may find use in the fabrication of spatially complex 3D scaffolds for tissue engineering applications, elucidation of the 3D structure-pharmacokinetic response relationship and the fabrication of complex multi-compartment liver tissue analog devices for drug screening applications.Mechanical Engineerin

Investigating Virus Clearance via pH Inactivation During Biomanufacturing

In the processing of biopharmaceuticals, viral clearance and viral safety are important for the development of monoclonal antibodies. Murine xenotropic leukemia virus (XMuLV) is one of the retroviruses, recommended by Food and Drug Administration (FDA) as a model virus for viral clearance via inactivation from therapeutics derived from Chinese hamster ovary cells (CHO). A robust and effective method was investigated to clear or inactivate endogenous viruses by low pH inactivation. The effects of different conductivity and inactivated time on XMuLV clearance was determined. Acetate buffer was prepared with different conductivity, and 2% XMuLV was spiked into acetate buffer. XMuLV virus particles could be effectively inactivated in acetate buffer at pH 3.6. According to TICD50 assay, the inactivation time of around 60 minutes was enough to clear all the viruses with more than 4 logs reduction value (LRV). Also, 50 mM acetate buffer has the most rapid inactivation process. TICD50 assays were able to determine the XMuLV virus titer within 95% confidence level, by using 8 replicates and 10-fold series dilution factor

Evaluation of electrospray differential mobility analysis for virus particle analysis: Potential applications for biomanufacturing.

The technique of electrospray differential mobility analysis (ES-DMA) was examined as a potential potency assay for routine virus particle analysis in biomanufacturing environments (e.g., evaluation of vaccines and gene delivery products for lot release) in the context of the International Committee of Harmonisation (ICH) Q2 guidelines. ES-DMA is a rapid particle sizing method capable of characterizing certain aspects of the structure (such as capsid proteins) and obtaining complete size distributions of viruses and virus-like particles. It was shown that ES-DMA can distinguish intact virus particles from degraded particles and measure the concentration of virus particles when calibrated with nanoparticles of known concentration. The technique has a measurement uncertainty of ≈20%, is linear over nearly 3 orders of magnitude, and has a lower limit of detection of ≈10(9)particles/mL. This quantitative assay was demonstrated for non-enveloped viruses. It is expected that ES-DMA will be a useful method for applications involving production and quality control of vaccines and gene therapy vectors for human use

Manufacturing Barriers to Biologics Competition and Innovation

As finding breakthrough small-molecule drugs gets harder, drug companies are increasingly turning to “large molecule” biologics. Although biologics represent many of the most promising new therapies for previously intractable diseases, they are extremely expensive. Moreover, the pathway for generic-type competition set up by Congress in 2010 is unlikely to yield significant cost savings. In this Article, we provide a fresh diagnosis of, and prescription for, this major public policy problem. We argue that the key cause is pervasive trade secrecy in the complex area of biologics manufacturing. Under the current regime, this trade secrecy, combined with certain features of FDA regulation, not only creates high barriers to entry of indefinite duration but also undermines efforts to advance fundamental knowledge. In sharp contrast, offering incentives for information disclosure to originator manufacturers would leverage the existing interaction of trade secrecy and the regulatory state in a positive direction. Although trade secrecy, particularly in complex areas like biologics manufacturing, often involves tacit knowledge that is difficult to codify and thus transfer, in this case regulatory requirements that originator manufacturers submit manufacturing details have already codified the relevant tacit knowledge. Incentivizing disclosure of these regulatory submissions would not only spur competition but it would provide a rich source of information upon which additional research, including fundamental research into the science of manufacturing, could build. In addition to provide fresh diagnosis and prescription in the specific area of biologics, the Article contributes to more general scholarship on trade secrecy and tacit knowledge. Prior scholarship has neglected the extent to which regulation can turn tacit knowledge not only into codified knowledge but into precisely the type of codified knowledge that is most likely to be useful and accurate. The Article also draws a link to the literature on adaptive regulation, arguing that greater regulatory flexibility is necessary and that more fundamental knowledge should spur flexibility. A vastly shortened version of the central argument that manufacturing trade secrecy hampers biosimilar development was published at 348 Science 188 (2015), available online

Design, Fabrication, and Testing of an Electrospinning Apparatus for the Deposition of PMMA Polymer for Biomedical Applications

This paper describes the successful design and fabrication of a deposition system for synthesis and assembly of nanoscale and submicron sized fibers of poly(methylmethacrylate)(PMMA) polymer. To optimize the electrospinning deposition process, the distance between the needle and the electrically grounded substrate, the applied voltage, and the concentration of PMMA polymer in the solution were varied. PMMA fibers as small as 500 nanometers were observed using scanning electron microscopy (SEM). The chemical signature of PMMA was confirmed for best quality and retention of chemistry using Fourier Transformed Infrared spectroscopy (FT-IR). PMMA is a biocompatible polymer, and nanofibers of PMMA are key building blocks for scaffolds and other biomanufacturing applications, such as bioprinting for regenerative medicine and tissue engineering of synthetic organs (Mo, 2004)

Synthetic biology and microbioreactor platforms for programmable production of biologics at the point-of-care

Current biopharmaceutical manufacturing systems are not compatible with portable or distributed production of biologics, as they typically require the development of single biologic-producing cell lines followed by their cultivation at very large scales. Therefore, it remains challenging to treat patients in short time frames, especially in remote locations with limited infrastructure. To overcome these barriers, we developed a platform using genetically engineered Pichia pastoris strains designed to secrete multiple proteins on programmable cues in an integrated, benchtop, millilitre-scale microfluidic device. We use this platform for rapid and switchable production of two biologics from a single yeast strain as specified by the operator. Our results demonstrate selectable and near-single-dose production of these biologics in <24 h with limited infrastructure requirements. We envision that combining this system with analytical, purification and polishing technologies could lead to a small-scale, portable and fully integrated personal biomanufacturing platform that could advance disease treatment at point-of-care

The Technology Innovation Program

[Excerpt] The Technology Innovation Program (TIP) at the National Institute of Standards and Technology (NIST) was established in 2007 to replace the Advanced Technology Program (ATP). This effort is designed to support, promote, and accelerate innovation in the United States through high-risk, high-reward research in areas of critical national need, according to the authorizing legislation. Grants are provided to small and medium-sized firms for individual projects or joint ventures with other research organizations. While similar to the Advanced Technology Program in the promotion of R&D that is expected to be of broad-based economic benefit to the nation, TIP appears to have been structured to avoid what was seen as government funding of large firms that opponents argued did not necessarily need federal support for research. The committee report to accompany H.R. 1868, part of which was incorporated into the final legislation, stated that TIP replaces ATP in consideration of a changing global innovation environment focusing on small and medium-sized companies. The design of the program also acknowledges the important role universities play in the innovation cycle by allowing universities to fully participate in the program. The elimination of ATP and the creation of TIP have renewed the debate over the role of the federal government in promoting commercial technology development. In arguing for less direct federal involvement, advocates of this approach believe that the market is superior to government in deciding technologies worthy of investment. Mechanisms that enhance the market\u27s opportunities and abilities to make such choices are preferred. It is suggested that agency discretion in selecting one technology over another can lead to political intrusion and industry dependency. On the other hand, supporters of direct methods argue that it is important to focus on those technologies that have the greatest promise as determined by industry and supported by matching funds from the private sector. They assert that the government can serve as a catalyst for cooperation As the Congress makes appropriation decisions, the discussion may serve to redefine thinking about governmental efforts in facilitating technological advancement in the private sector

Automating functional enzyme screening & characterization

This work has been presented in the 10th IWBDA workshop.Microfluidics continue to gain traction as an inexpensive alternative to standard multi-well plate-based, and flow cytometry- based, assay platforms. These devices are especially useful for the types of ultra-high throughput screens needed for enzyme discovery applications where large numbers (>106) of unique samples must be screened rapidly1. Coupled with cell-free protein synthesis2, microfluidics are being used to identify novel enzymes useful for a variety of applications with unprecedented speed. However, these devices are typically produced using PDMS, and require considerable infrastructure and artisanal skill to fabricate, limiting their accessibility. Likewise, enzyme hits obtained from a screen are often validated manually and would benefit from automation of downstream validation processes. To address these limitations, we propose a workflow which leverages software tools to automate the rapid design and fabrication of low-cost polycarbonate microfluidic devices for use as high-throughput screening platforms for enzyme discovery, as well as an automated DNA assembly tool to streamline validation of screening candidates. Using this workflow, we aim to identify novel oxidoreductase enzymes from environmental metagenomic DNA libraries, for use in electrochemical biosensors