Democratizing global supply of recombinant proteins

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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

Accelerated process development for integrated end-to-end biologics manufacturing

With the exception of monoclonal antibodies, biologics typically require bespoke manufacturing processes that vary widely in the type of and number of unit operations. This constraint leads to custom facility designs and unique strategies for process development for every new molecule. To enable flexible, multi-product manufacturing facilities and to reduce the speed to clinic for new molecules, streamlined manufacturing processes and associated strategies for process development are needed. We have developed a bench-scale, integrated and automated manufacturing platform capable of rapidly producing a variety of recombinant proteins with phase-appropriate quality for early development1. The system comprises three modules for fermentation via perfusion, straight-through chromatographic purification, and formulation. To facilitate the production of multiple products on the same system, we have also developed a holistic strategy for process design to manufacture new products in as few as twelve weeks after obtaining the product sequence. While upstream process development in our host (Pichia pastoris) has been relatively straightforward, there are not many tools currently available for developing fully integrated straight-through chromatographic processes. Therefore, we developed an in silico tool for the prediction of fully integrated purification processes based on a one-time collection of host-related data combined with conventional high-throughput chromatographic screening data for each new target molecule2. We used this tool to develop fully integrated, end-to-end production processes for three molecules (hGH, IFNα-2b, and G-CSF) with at least 45% fewer steps than traditional processes. While our in silico tool allows for rapid resin selection, it may not predict the optimal process for each individual molecule since it is based on conventional high-throughput screening techniques which seek to optimize each chromatographic step independently rather than optimizing a fully integrated, multi-column process. To address this limitation, we have also developed a DoE-like framework for the optimization of fully integrated purification processes once the resins have been selected. First, a series of range finding experiments are carried out on each individual column, similar to conventional screening but with limited analytics. Next, we carry out fully integrated (multi-column) testing of the proposed operational area with more extensive analytics, including host cell protein, DNA, and yield measurements. We use this methodology to develop optimized processes for the end-to-end production of a variety of single domain antibodies with high yield and purity. Further, we present a method for predicting the optimal operating conditions for a new molecule within the same class based only on its biophysical characteristics, reducing the timeline from sequence to early stage, phase-appropriate product to only six weeks. Using these holistic strategies for process development, we have produced over ten different recombinant proteins on our manufacturing platform including enzymes, cytokines, singe domain antibodies, and vaccine subunits. We believe that such integrated strategies for process design could enable the rapid translation from sequence to early stage clinical development of products for a variety of molecules and potentially allow clinical testing of a greater number of high quality molecules for vaccines and biopharmaceuticals. 1. Crowell, L. E. et al. On-demand manufacturing of clinical-quality biopharmaceuticals. Nat. Biotechnol. (2018). doi:10.1038/nbt.4262 2. Timmick, S. M. et al. An impurity characterization based approach for the rapid development of integrated downstream purification processes. Biotechnol. Bioeng. 1–13 (2018). doi:10.1002/bit.2671

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

Designing a microbial cultivation platform for continuous biopharmaceutical production

The existing biopharmaceutical manufacturing paradigm is poorly suited to produce biologic drugs on demand at a point-of-care. Generally, commercial-scale (~2,000 - 10,000 L) manufacturing using fed-batch cultivation and fixed stainless-steel infrastructure is concentrated in developed nations and results in process cycle times on the order of weeks to months.1,2 Coupled with the complex logistical challenges associated with continuous “plant-to-patient” cold-chains, the geographically biased nature of therapeutic protein production today can limit access to biologic drugs in developing areas of the world.3 There is an opportunity to create technologies capable of rapidly generating biopharmaceuticals in situ in emergency situations, in remote healthcare settings, and in the battlefield. A platform that incorporates a modular suite of bioreactor, purification, and in-line analytics technologies has the potential to bridge this gap if developed in parallel with appropriately engineered stains of a flexible expression host. This poster will describe a multifaceted approach towards the development of a fully automated bench-scale perfusion process for the cultivation of Pichia pastoris and expression of therapeutically relevant heterologous proteins. We demonstrate the application of computational fluid dynamics (CFD) simulations to the optimization of the cultivation environment within our bench-top bioreactors. We further show that Pichia pastoris is amenable to secreting a variety of recombinant proteins spanning a range of preexisting drug classes (e.g. hormones, cytokines, monoclonal antibodies, vaccine antigens). Among these therapeutic proteins are molecules that require proper co-/post-translational processing for bioactivity. We envision that the development of P. pastoris strains with the capability to perform these critical processing steps in vivo will mitigate the need to chemically modify proteins post-expression and reduce the number of unit operations required in a typical upstream process

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

A review of consumer awareness, understanding and use of food-based dietary guidelines

Copyright @ 2011 Cambridge University PressFood-based dietary guidelines (FBDG) have primarily been designed for the consumer to encourage healthy, habitual food choices, decrease chronic disease risk and improve public health. However, minimal research has been conducted to evaluate whether FBDG are utilised by the public. The present review used a framework of three concepts, awareness, understanding and use, to summarise consumer evidence related to national FBDG and food guides. Searches of nine electronic databases, reference lists and Internet grey literature elicited 939 articles. Predetermined exclusion criteria selected twenty-eight studies for review. These consisted of qualitative, quantitative and mixed study designs, non-clinical participants, related to official FBDG for the general public, and involved measures of consumer awareness, understanding or use of FBDG. The three concepts of awareness, understanding and use were often discussed interchangeably. Nevertheless, a greater amount of evidence for consumer awareness and understanding was reported than consumer use of FBDG. The twenty-eight studies varied in terms of aim, design and method. Study quality also varied with raw qualitative data, and quantitative method details were often omitted. Thus, the reliability and validity of these review findings may be limited. Further research is required to evaluate the efficacy of FBDG as a public health promotion tool. If the purpose of FBDG is to evoke consumer behaviour change, then the framework of consumer awareness, understanding and use of FBDG may be useful to categorise consumer behaviour studies and complement the dietary survey and health outcome data in the process of FBDG evaluation and revision.This study is funded by the European Commission Sixth Framework Programme (contract no. 036196)