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

Systematic Single-Cell Analysis of Pichia pastoris Reveals Secretory Capacity Limits Productivity

Biopharmaceuticals represent the fastest growing sector of the global pharmaceutical industry. Cost-efficient production of these biologic drugs requires a robust host organism for generating high titers of protein during fermentation. Understanding key cellular processes that limit protein production and secretion is, therefore, essential for rational strain engineering. Here, with single-cell resolution, we systematically analysed the productivity of a series of Pichia pastoris strains that produce different proteins both constitutively and inducibly. We characterized each strain by qPCR, RT-qPCR, microengraving, and imaging cytometry. We then developed a simple mathematical model describing the flux of folded protein through the ER. This combination of single-cell measurements and computational modelling shows that protein trafficking through the secretory machinery is often the rate-limiting step in single-cell production, and strategies to enhance the overall capacity of protein secretion within hosts for the production of heterologous proteins may improve productivity

Automated synthesis of the Lewis blood group oligosaccharides

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2004.Vita.Includes bibliographical references.Cell-surface carbohydrates are markers of specific cell types. These oligosaccharides are involved in recognition, adhesion, and signal transduction events. Advances in molecular glycobiology rely heavily on straightforward access to structurally defined oligosaccharides, but traditional syntheses of complex carbohydrates have been very laborious. Development of a novel linker and monitoring of each glycosylation reaction during automated solid-phase oligosaccharide synthesis allowed for the rapid synthesis of three Lewis-type cell surface oligosaccharides. The assembly of the nonasaccharide adenocarcinoma marker Le[superscript]y-Le[superscript]x monosaccharide building blocks was achieved in just 23 hours, while the syntheses of the tumor markers Lewis X, a pentasaccharide, and Lewis Y, a hexasaccharide, required only 12 and 14 hours respectively. The automation of carbohydrate synthesis greatly accelerates access to molecules for biological study and vaccine development.by Kerry Routenberg Love.Ph.D

Immuno-Hybridization Chain Reaction for Enhancing Detection of Individual Cytokine-Secreting Human Peripheral Mononuclear Cells

We present here a new method to enhance the detection of secreted cytokines and chemokines from single human mononuclear cells. The technique uses a hybridization chain reaction (HCR) to amplify signals resulting from sandwich immunoassays. This immuno-HCR employs oligonucleotide-based initiators covalently linked to antibodies to propagate a chain reaction of hybridization events involving a pair of complementary hairpin oligomers bearing fluorescent labels. Integrating this strategy for signal amplification with microengraving (a soft lithographic method for printing arrays of secreted proteins from thousands of single cells) improves both the limits of detection and sensitivity for cytokines and chemokines captured from individual cells by an average of 200-fold relative to methods for direct detection by fluoresence. This approach should enhance the utility of microengraving for defining the immunological signatures of diseases and responses to interventional therapies based on multiplexed single-cell analysis. © 2011 American Chemical Society

Identifying Improved Sites for Heterologous Gene Integration Using ATAC-seq

Copyright © 2020 American Chemical Society. Constructing efficient cellular factories often requires integration of heterologous pathways for synthesis of novel compounds and improved cellular productivity. Few genomic sites are routinely used, however, for efficient integration and expression of heterologous genes, especially in nonmodel hosts. Here, a data-guided framework for informing suitable integration sites for heterologous genes based on ATAC-seq was developed in the nonmodel yeast Komagataella phaffii. Single-copy GFP constructs were integrated using CRISPR/Cas9 into 38 intergenic regions (IGRs) to evaluate the effects of IGR size, intensity of ATAC-seq peaks, and orientation and expression of adjacent genes. Only the intensity of accessibility peaks was observed to have a significant effect, with higher expression observed from IGRs with low-to moderate-intensity peaks than from high-intensity peaks. This effect diminished for tandem, multicopy integrations, suggesting that the additional copies of exogenous sequence buffered the transcriptional unit of the transgene against effects from endogenous sequence context. The approach developed from these results should provide a basis for nominating suitable IGRs in other eukaryotic hosts from an annotated genome and ATAC-seq data

Identifying Improved Sites for Heterologous Gene Integration Using ATAC-seq

Copyright © 2020 American Chemical Society. Constructing efficient cellular factories often requires integration of heterologous pathways for synthesis of novel compounds and improved cellular productivity. Few genomic sites are routinely used, however, for efficient integration and expression of heterologous genes, especially in nonmodel hosts. Here, a data-guided framework for informing suitable integration sites for heterologous genes based on ATAC-seq was developed in the nonmodel yeast Komagataella phaffii. Single-copy GFP constructs were integrated using CRISPR/Cas9 into 38 intergenic regions (IGRs) to evaluate the effects of IGR size, intensity of ATAC-seq peaks, and orientation and expression of adjacent genes. Only the intensity of accessibility peaks was observed to have a significant effect, with higher expression observed from IGRs with low-to moderate-intensity peaks than from high-intensity peaks. This effect diminished for tandem, multicopy integrations, suggesting that the additional copies of exogenous sequence buffered the transcriptional unit of the transgene against effects from endogenous sequence context. The approach developed from these results should provide a basis for nominating suitable IGRs in other eukaryotic hosts from an annotated genome and ATAC-seq data

Comparative genome‐scale analysis of Pichia pastoris variants informs selection of an optimal base strain

© 2019 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc. Komagataella phaffii, also known as Pichia pastoris, is a common host for the production of biologics and enzymes, due to fast growth, high productivity, and advancements in host engineering. Several K. phaffii variants are commonly used as interchangeable base strains, which confounds efforts to improve this host. In this study, genomic and transcriptomic analyses of Y-11430 (CBS7435), GS115, X-33, and eight other variants enabled a comparative assessment of the relative fitness of these hosts for recombinant protein expression. Cell wall integrity explained the majority of the variation among strains, impacting transformation efficiency, growth, methanol metabolism, and secretion of heterologous proteins. Y-11430 exhibited the highest activity of genes involved in methanol utilization, up to two-fold higher transcription of heterologous genes, and robust growth. With a more permeable cell wall, X-33 displayed a six-fold higher transformation efficiency and up to 1.2-fold higher titers than Y-11430. X-33 also shared nearly all mutations, and a defective variant of HIS4, with GS115, precluding robust growth. Transferring two beneficial mutations identified in X-33 into Y-11430 resulted in an optimized base strain that provided up to four-fold higher transformation efficiency and three-fold higher protein titers, while retaining robust growth. The approach employed here to assess unique banked variants in a species and then transfer key beneficial variants into a base strain should also facilitate rational assessment of a broad set of other recombinant hosts

Analysis of steady-state distributions of rates of secretion.

<p>(A) Distributions of rates of secretion of eGFP for (Top) a clone with a single copy of eGFP under transcriptional control of pGAPDH, and (Bottom) a clone with two copies of eGFP under transcriptional control of pAOX1. Red squares indicate binned single-cell secretion events following microengraving with each clone. Blue lines show the best fits using Eq. (1). Values for <i>a</i> and <i>b</i> are shown. (B) Relationship between <i>a</i> (burst frequency) and <i>b</i> (burst size) for proteins expressed using either pGAPDH (top) or pAOX1 (bottom) as a function of gene copy number and complexity. Clones secreting eGFP (green), clones secreting aglycosylated Fc fragment (blue) and clones secreting glycosylated Fc fragment (red) are shown for a single gene copy (squares), 2–3 gene copies (triangles) and 4 or more gene copies (circles). Error bars represent S.E.M. for each clone from at least three separate measurements.</p

<i>Pichia pastoris</i> strains generated for systematic investigation of the relationship between protein complexity, gene dosage, relative expression and secretion.

a<p>Strain names indicate promoter used.</p>b<p>Determined by qPCR using an absolute quantification of transcript copy number.</p>c<p>Determined by RT-qPCR using an absolute quantification of transcript copy number and the expression of actin as a control.</p