Integrated scalable cyto-technology for recombinant protein bioprocessing

Biological knowledge of infectious diseases and other diseases for which vaccines may provide therapeutic benefits, such as cancer, is growing at an accelerated pace. The implications of this knowledge are improved stratification of diseases, possibilities for personalized treatments, and explicit understanding of protective immune responses to be elicited by vaccines. With this knowledge, it is becoming increasingly feasible to engineer vaccines for specific responses rather than relying on empirical development. Despite this potential, the challenge of routine, low-cost manufacturing of vaccines creates a barrier to transforming health care in both high- and low-resource countries. Vaccines today do not benefit from well-defined, platform-like processes for manufacturing, and concepts such as continuous bioprocessing remain largely within the realm of biopharmaceutical products. The InSCyT platform is an advanced prototype manufacturing system that provides integrated and automated production and purification of multiple protein therapeutics. The system allows end-to-end manufacturing of 100\u27s to 10,000\u27s of doses of recombinant protein drugs in days. It uses a state-of-the-art approach to process design and implementation that takes advantage of a fast-growing, tractable microbial host (Pichia pastoris) and continuous processing for automated, hands-free purification through simple 2- or 3-stage chromatographic processes. The platform design is highly modular, allowing facile process development and process deployment for multiple products. This feature emerges from the predictable behavior of the fermentation and cell culture fluids, and rapid cloning of new molecules, that together facilitate fast development of entirely new processes in weeks. To date, this system has been used to reproducibly manufacture high-quality human growth hormone (hGH), granulocyte-colony stimulating factor (G-CSF), and interferon-α2b (IFN-α2b) in an integrated, automated manner. The speed of production using the InSCyT prototype allows volumetric productivities that compare favorably to those for mammalian-based production. This talk will outline the design and capabilities of the InSCyT system, demonstrate the quality of biologic drugs made to date on the system, and outline opportunities for advancing the platform to provide new capabilities in manufacturing recombinant proteins for use in vaccines. As part of a Gates Foundation-funded Grand Challenge called ULTRA, we have begun to assess the feasibility of manufacturing millions of doses of a trivalent recombinant rotavirus vaccine annually on a small-scale production system like InSCyT. Integrated bioprocessing enabled by systems such as these could offer potential advantages for routine production in local regions with minimal infrastructure, and for democratization of manufacturing capacity for new vaccine concepts and personalized treatments in cancer

Rational design of expression vectors for high quality biologics

Commercial proteins (e.g. antibodies, enzymes, vaccine components) for applications from biopharmaceuticals to commodity chemicals require low-cost manufacturing of high-quality products. The engineering of recombinant hosts to achieve large quantities of high-quality heterologous proteins is crucial to both minimizing costs and maximizing safety and efficacy (in the case of biopharmaceuticals). High-quality proteins are properly folded and full-length (intact), with native N-, and C-, termini and bear no significant proteolysis or other degradation (oxidation, deamidation, etc…). As most expression hosts rely on recombinant DNA technology for production of the heterologous protein, the transgene cassette provides an early, and inexpensive, opportunity for optimization of quality and protein titer. Commonly, transgene cassettes include a promoter, a heterologous gene of interest, and terminator for expression of the heterologous gene. A targeting sequence for guided recombination and selective marker for isolation of positive clones are also key elements. In engineering the transgene cassette, factors such as the promoter for heterologous gene expression, target site for transgene integration, sequence for translation initiation, and mRNA codon-optimization of the gene of interest are critical design points for a given protein-expressing strain. Here, we demonstrate an approach to transgene cassette optimization in the methylotrophic yeast, Pichia pastoris, informed by functional genomics. Omics-based techniques such as RNA-Seq, ATAC-Seq and ribosomal foot-printing afford greater upfront understanding for subsequent optimized strain engineering on a product-by-product basis. These types of data are cheap and easy to acquire for yeast and can indicate host- or sequence-derived bottlenecks in transgene transcription, translation and expression. Linking these data to product quality attributes can enlighten the design of the expression vector for fast in silico optimization of wide-ranging factors such as gene dosage balance, translation efficiency, and balanced cell kinetics enabling high-quality protein production. Collectively, we show that these tools can enable fast vector design for new heterologous protein-producing strains, including those expressing recombinant vaccines, and robust optimization when engineering higher productivity cell lines

Molecular quality engineering for low cost vaccine production

Vaccines based on recombinant proteins provide a compelling case for low cost products with broad global accessibility. Protein immunogens are typically derived directly from native sequences found in bacterial or viral pathogens, and may not be well-suited for efficient expression in recombinant hosts. Native immunogens may also suffer from numerous challenges during expression that impact their quality or efficient production, including truncation, aggregation and poor stability. These challenges can lead to inefficiencies in manufacturing of subunit protein vaccines. Typically, recombinant vaccine manufacturing processes are complex, serial batch operations requiring extensive quality testing throughout to ensure product integrity. In response to the Gates Foundation’s Grand Challenge for Innovations in Vaccine Manufacturing for Global Markets, we are co-developing the ULTRA program for flexible, low cost vaccine products. This program aims to develop platform processes for production of recombinant vaccines. We believe that molecular design of the antigens provides a critical handle in improving antigen quality, manufacturability, and product stability, all of which could enable potent, low-cost vaccines. Addressing potential manufacturing challenges early on in product development should enable simple integrated processes for antigen production while minimizing costs associated with quality testing. To this end, we are demonstrating our platform approach with a recombinant trivalent subunit vaccine for rotavirus currently in clinical development. We chose to express the three VP8 subunits in Pichia pastoris to take advantage of the high titers of secreted proteins and minimal process-related contaminants typically experienced with this organism—critical features when developing simple intensified processes to meet our cost targets of $0.15/dose. Initial expression results showed the rotavirus antigens were poorly expressed and suffered from N-terminal truncation and aggregation—all of which were also observed in a previously developed E. coli-based process. We have deployed a two-pronged approach toward improving the manufacturability of these antigens. First, we used a functional genomics approach to identify bottlenecks experienced during cellular expression of the antigens. RNA-sequencing is a mature, inexpensive and acccessible technique for yeast that can indicate host- or sequence-derived bottlenecks in antigen transcription, translation and expression. Second, we made direct sequence changes to the antigens to mitigate specific quality challenges, such as aggregation. Iterations of this approach have enabled robust titers of rotavirus antigens with improved quality. This framework for incorporation of molecular engineering early in development provides a useful model for improving target product profiles that include manufacturability for low-costs, while maintaining immunogenicity

Identifying the best Pichia pastoris base strain using functional genomics

Market sizes for novel breakthrough therapies and growing demand for existing treatments in emerging markets promise to challenge the current capacity for production of biologics. These trends dictate the need for a concomitant paradigm shift in biomanufacturing toward greater productivity for lower cost. Strain engineering is a promising means to realize the greatest returns by increasing the product titer going into downstream processes. Current cellular hosts are approaching saturation of optimal productivity due to lack of deep biological understanding or limitations of the host’s intrinsic secretion capacity. We demonstrate an approach informed by functional genomics to understand key performance differences between interchangeably-used variants of the host, Pichia pastoris. Genomic variant calling on all USDA-banked and commercially-available strains revealed varying numbers of SNPs relative to the WT strain, Y-11430. Combining transcriptomics and traditional phenotypic assays, the functional impact of these SNPs can inform which host strain is best suited for a given application. Taken together, we have identified key, beneficial SNPs that can be introduced into a WT background to create an IP-free host primed for optimal protein production

A perfusion-capable microfluidic bioreactor for assessing microbial heterologous protein production

We present an integrated microfluidic bioreactor for fully continuous perfusion cultivation of suspended microbial cell cultures. This system allowed continuous and stable heterologous protein expression by sustaining the cultivation of Pichia pastoris over 11 days. This technical capability also allowed testing the impact of perfusion conditions on protein expression. This advance should enable small-scale models for process optimization in continuous biomanufacturing.United States. Defense Advanced Research Projects Agency (N66001-13-C-4025)National Cancer Institute (U.S.) (P30-CA14051)United States. National Institutes of Health (2T32GM008334-26

Automated pipeline for rapid production and screening of HIV-specific monoclonal antibodies using pichia pastoris

Monoclonal antibodies (mAbs) that bind and neutralize human pathogens have great therapeutic potential. Advances in automated screening and liquid handling have resulted in the ability to discover antigen-specific antibodies either directly from human blood or from various combinatorial libraries (phage, bacteria or yeast). There remain, however, bottlenecks in the cloning, expression and evaluation of such lead antibodies identified in primary screens that hinder high-throughput screening. As such, ‘hit-to-lead identification’ remains both expensive and time-consuming. By combining the advantages of overlap extension PCR (OE-PCR) and a genetically stable yet easily manipulatable microbial expression host Pichia pastoris, we have developed an automated pipeline for the rapid production and screening of full-length antigenspecific mAbs. Here, we demonstrate the speed, feasibility and cost-effectiveness of our approach by generating several broadly neutralizing antibodies against human immunodeficiency virus (HIV).Bill & Melinda Gates FoundationUnited States. Defense Advanced Research Projects AgencySpace and Naval Warfare Systems Center San Diego (U.S.) (Contract N66001-13-C-4025)W. M. Keck FoundationNational Institute of Allergy and Infectious Diseases (U.S.) (U19AI090970).National Cancer Institute (U.S.) (David H. Koch Institute for Integrative Cancer Research at MIT. Support (Core) Grant P30-CA14051

Molecular engineering improves antigen quality and enables integrated manufacturing of a trivalent subunit vaccine candidate for rotavirus

Background Vaccines comprising recombinant subunit proteins are well-suited to low-cost and high-volume production for global use. The design of manufacturing processes to produce subunit vaccines depends, however, on the inherent biophysical traits presented by an individual antigen of interest. New candidate antigens typically require developing custom processes for each one and may require unique steps to ensure sufficient yields without product-related variants. Results We describe a holistic approach for the molecular design of recombinant protein antigens—considering both their manufacturability and antigenicity—informed by bioinformatic analyses such as RNA-seq, ribosome profiling, and sequence-based prediction tools. We demonstrate this approach by engineering the product sequences of a trivalent non-replicating rotavirus vaccine (NRRV) candidate to improve titers and mitigate product variants caused by N-terminal truncation, hypermannosylation, and aggregation. The three engineered NRRV antigens retained their original antigenicity and immunogenicity, while their improved manufacturability enabled concomitant production and purification of all three serotypes in a single, end-to-end perfusion-based process using the biotechnical yeast Komagataella phaffii. Conclusions This study demonstrates that molecular engineering of subunit antigens using advanced genomic methods can facilitate their manufacturing in continuous production. Such capabilities have potential to lower the cost and volumetric requirements in manufacturing vaccines based on recombinant protein subunits

NACHOS, a CubeSat-Based High-Resolution UV-Visible Hyperspectral Imager for Remote Sensing of Trace Gases: System Overview, Science Objectives, and Preliminary Results

The Nano-satellite Atmospheric Chemistry Hyperspectral Observation System (NACHOS) is a high-throughput (f/2.9), high spectral resolution (1.3 nm optical, 0.57 nm sampling) hyperspectral imager covering the 300-500 nm spectral region with 350 spectral bands. The combined 1.5U instrument payload and 1.5U spacecraft bus comprise a 3U CubeSat. Spectroscopically similar to NASA’s Ozone Monitoring Instrument (OMI), which provides wide-field coverage at ~20 km spatial resolution, NACHOS offers complementary targeted measurements at far higher spatial resolution of ~0.4 km/pixel from 500 km altitude over its 15 ̊ across-track field of view. NACHOS incorporates highly streamlined onboard gas-retrieval algorithms, alleviating the need to routinely downlink massive hyperspectral data cubes. This paper discusses the instrument design, requirements leading to it, preliminary results, and science goals, including monitoring NO2 as a proxy for anthropogenic greenhouse gases, low-level degassing of SO2 and halogen oxides at pre-eruptive volcanoes, and formaldehyde from wildfires. Aiming for an eventual many-satellite constellation providing both high spatial resolution and frequent target revisits, the current NACHOS project is launching two CubeSats, the first already launched to the International Space Station aboard the NG-17 Cygnus vehicle on February 19, 2022 and awaiting deployment to its final orbit in June, and the second launching June 29, 2022

The James Webb Space Telescope Mission

Twenty-six years ago a small committee report, building on earlier studies, expounded a compelling and poetic vision for the future of astronomy, calling for an infrared-optimized space telescope with an aperture of at least $4m$. With the support of their governments in the US, Europe, and Canada, 20,000 people realized that vision as the $6.5m$ James Webb Space Telescope. A generation of astronomers will celebrate their accomplishments for the life of the mission, potentially as long as 20 years, and beyond. This report and the scientific discoveries that follow are extended thank-you notes to the 20,000 team members. The telescope is working perfectly, with much better image quality than expected. In this and accompanying papers, we give a brief history, describe the observatory, outline its objectives and current observing program, and discuss the inventions and people who made it possible. We cite detailed reports on the design and the measured performance on orbit.Comment: Accepted by PASP for the special issue on The James Webb Space Telescope Overview, 29 pages, 4 figure

Search for dark matter produced in association with bottom or top quarks in √s = 13 TeV pp collisions with the ATLAS detector

A search for weakly interacting massive particle dark matter produced in association with bottom or top quarks is presented. Final states containing third-generation quarks and miss- ing transverse momentum are considered. The analysis uses 36.1 fb−1 of proton–proton collision data recorded by the ATLAS experiment at √s = 13 TeV in 2015 and 2016. No significant excess of events above the estimated backgrounds is observed. The results are in- terpreted in the framework of simplified models of spin-0 dark-matter mediators. For colour- neutral spin-0 mediators produced in association with top quarks and decaying into a pair of dark-matter particles, mediator masses below 50 GeV are excluded assuming a dark-matter candidate mass of 1 GeV and unitary couplings. For scalar and pseudoscalar mediators produced in association with bottom quarks, the search sets limits on the production cross- section of 300 times the predicted rate for mediators with masses between 10 and 50 GeV and assuming a dark-matter mass of 1 GeV and unitary coupling. Constraints on colour- charged scalar simplified models are also presented. Assuming a dark-matter particle mass of 35 GeV, mediator particles with mass below 1.1 TeV are excluded for couplings yielding a dark-matter relic density consistent with measurements