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1.
Proc Natl Acad Sci U S A ; 118(38)2021 09 21.
Artículo en Inglés | MEDLINE | ID: mdl-34493582

RESUMEN

Global containment of COVID-19 still requires accessible and affordable vaccines for low- and middle-income countries (LMICs). Recently approved vaccines provide needed interventions, albeit at prices that may limit their global access. Subunit vaccines based on recombinant proteins are suited for large-volume microbial manufacturing to yield billions of doses annually, minimizing their manufacturing cost. These types of vaccines are well-established, proven interventions with multiple safe and efficacious commercial examples. Many vaccine candidates of this type for SARS-CoV-2 rely on sequences containing the receptor-binding domain (RBD), which mediates viral entry to cells via ACE2. Here we report an engineered sequence variant of RBD that exhibits high-yield manufacturability, high-affinity binding to ACE2, and enhanced immunogenicity after a single dose in mice compared to the Wuhan-Hu-1 variant used in current vaccines. Antibodies raised against the engineered protein exhibited heterotypic binding to the RBD from two recently reported SARS-CoV-2 variants of concern (501Y.V1/V2). Presentation of the engineered RBD on a designed virus-like particle (VLP) also reduced weight loss in hamsters upon viral challenge.


Asunto(s)
Vacunas contra la COVID-19/inmunología , COVID-19/prevención & control , Ingeniería de Proteínas/métodos , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/genética , Animales , Anticuerpos Antivirales/inmunología , Antígenos Virales , Sitios de Unión , COVID-19/virología , Vacunas contra la COVID-19/economía , Humanos , Inmunogenicidad Vacunal , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Unión Proteica , Conformación Proteica , Saccharomycetales/metabolismo , Vacunas de Subunidad
2.
Biotechnol Bioeng ; 118(5): 1832-1839, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33527350

RESUMEN

Development of continuous biopharmaceutical manufacturing processes is an area of active research. This study considers the long-term transgene copy number stability of Pichia pastoris in continuous bioreactors. We propose a model of copy number loss that quantifies population heterogeneity. An analytical solution is derived and compared with existing experimental data. The model is then used to provide guidance for stable operating timescales. The model is extended to consider copy number dependent growth such as in the case of Zeocin supplementation. The model is also extended to analyze a continuous seeding strategy. This study is a critical step towards understanding the impact of continuous processing on the stability of Pichia pastoris and the resultant products.


Asunto(s)
Reactores Biológicos/microbiología , Variaciones en el Número de Copia de ADN/genética , Inestabilidad Genómica/genética , Proteínas Recombinantes , Saccharomycetales , ADN de Hongos/genética , Modelos Genéticos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo
3.
Microb Cell Fact ; 20(1): 94, 2021 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-33933073

RESUMEN

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.


Asunto(s)
Antígenos Virales/genética , Ingeniería Genética/métodos , Vacunas contra Rotavirus/genética , Rotavirus/inmunología , Saccharomycetales/genética , Antígenos Virales/inmunología , Biología Computacional , Genómica/métodos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Rotavirus/genética , Vacunas contra Rotavirus/inmunología , Vacunas de Subunidad/genética , Vacunas de Subunidad/inmunología
4.
Biotechnol Bioeng ; 117(2): 543-555, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-31654411

RESUMEN

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.


Asunto(s)
Proteínas Fúngicas/genética , Genoma Fúngico/genética , Pichia/genética , Proteínas Recombinantes/genética , Transcriptoma/genética , Proteínas Fúngicas/metabolismo , Perfilación de la Expresión Génica , Genómica , Pichia/metabolismo , ARN de Hongos/análisis , ARN de Hongos/genética , Proteínas Recombinantes/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Análisis de Secuencia de ARN
5.
J Pharm Sci ; 110(3): 1054-1066, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33278412

RESUMEN

In a companion paper, a two-step developability assessment is presented to rapidly evaluate low-cost formulations (multi-dose, aluminum-adjuvanted) for new subunit vaccine candidates. As a case study, a non-replicating rotavirus (NRRV) recombinant protein antigen P[4] was found to be destabilized by the vaccine preservative thimerosal, and this effect was mitigated by modification of the free cysteine (C173S). In this work, the mechanism(s) of thimerosal-P[4] protein interactions, along with subsequent effects on the P[4] protein's structural integrity, are determined. Reversible complexation of ethylmercury, a thimerosal degradation byproduct, with the single cysteine residue of P[4] protein is demonstrated by intact protein mass analysis and biophysical studies. A working mechanism involving a reversible S-Hg coordinate bond is presented based on the literature. This reaction increased the local backbone flexibility of P[4] within the helical region surrounding the cysteine residue and then caused more global destabilization, both as detected by HX-MS. These effects correlate with changes in antibody-P[4] binding parameters and alterations in P[4] conformational stability due to C173S modification. Epitope mapping by HX-MS demonstrated involvement of the same cysteine-containing helical region of P[4] in antibody-antigen binding. Future formulation challenges to develop low-cost, multi-dose formulations for new recombinant protein vaccine candidates are discussed.


Asunto(s)
Rotavirus , Timerosal , Antígenos Virales , Conservadores Farmacéuticos , Vacunas de Subunidad
6.
J Pharm Sci ; 110(3): 1042-1053, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33285182

RESUMEN

A two-step developability assessment workflow is described to screen variants of recombinant protein antigens under various formulation conditions to rapidly identify stable, aluminum-adjuvanted, multi-dose vaccine candidates. For proof-of-concept, a series of sequence variants of the recombinant non-replicating rotavirus (NRRV) P[8] protein antigen (produced in Komagataella phaffii) were compared in terms of primary structure, post-translational modifications, antibody binding, conformational stability, relative solubility and preservative compatibility. Based on these results, promising P[8] variants were down-selected and the impact of key formulation conditions on storage stability was examined (e.g., presence or absence of the aluminum-adjuvant Alhydrogel and the preservative thimerosal) as measured by differential scanning calorimetry (DSC) and antibody binding assays. Good correlations between rapidly-generated developability screening data and storage stability profiles (12 weeks at various temperatures) were observed for aluminum-adsorbed P[8] antigens. These findings were extended and confirmed using variants of a second NRRV antigen, P[4]. These case-study results with P[8] and P[4] NRRV variants are discussed in terms of using this vaccine formulation developability workflow to better inform and optimize formulation design with a wide variety of recombinant protein antigens, with the long-term goal of rapidly and cost-efficiently identifying low-cost vaccine formulations for use in low and middle income countries.


Asunto(s)
Vacunas contra Rotavirus , Rotavirus , Antígenos , Proteínas Recombinantes , Saccharomycetales
7.
bioRxiv ; 2021 Mar 04.
Artículo en Inglés | MEDLINE | ID: mdl-33688647

RESUMEN

Global containment of COVID-19 still requires accessible and affordable vaccines for low- and middle-income countries (LMICs).1 Recently approved vaccines provide needed interventions, albeit at prices that may limit their global access.2 Subunit vaccines based on recombinant proteins are suited for large-volume microbial manufacturing to yield billions of doses annually, minimizing their manufacturing costs.3 These types of vaccines are well-established, proven interventions with multiple safe and efficacious commercial examples.4-6 Many vaccine candidates of this type for SARS-CoV-2 rely on sequences containing the receptor-binding domain (RBD), which mediates viral entry to cells via ACE2.7,8 Here we report an engineered sequence variant of RBD that exhibits high-yield manufacturability, high-affinity binding to ACE2, and enhanced immunogenicity after a single dose in mice compared to the Wuhan-Hu-1 variant used in current vaccines. Antibodies raised against the engineered protein exhibited heterotypic binding to the RBD from two recently reported SARS-CoV-2 variants of concern (501Y.V1/V2). Presentation of the engineered RBD on a designed virus-like particle (VLP) also reduced weight loss in hamsters upon viral challenge.

8.
ACS Synth Biol ; 9(1): 26-35, 2020 01 17.
Artículo en Inglés | MEDLINE | ID: mdl-31825599

RESUMEN

There is growing interest in the use of nonmodel microorganisms as hosts for biopharmaceutical manufacturing. These hosts require genomic engineering to meet clinically relevant product qualities and titers, but the adaptation of tools for editing genomes, such as CRISPR-Cas9, has been slow for poorly characterized hosts. Specifically, a lack of biochemical characterization of RNA polymerase III transcription has hindered reliable expression of guide RNAs in new hosts. Here, we present a sequencing-based strategy for the design of host-specific cassettes for modular, reliable, expression of guide RNAs. Using this strategy, we achieved up to 95% gene editing efficiency in the methylotrophic yeast Komagataella phaffii. We applied this approach for the rapid, multiplexed engineering of a complex phenotype, achieving humanized product glycosylation in two sequential steps of engineering. Reliable extension of simple gene editing tools to nonmodel manufacturing hosts will enable rapid engineering of manufacturing strains tuned for specific product profiles and potentially decrease the costs and timelines for process development.


Asunto(s)
Sistemas CRISPR-Cas , Edición Génica/métodos , Genoma Fúngico , ARN Guía de Kinetoplastida/genética , Saccharomycetales/genética , Regiones no Traducidas 3' , Secuencia de Bases , Productos Biológicos , Proteínas Fúngicas/genética , Técnicas de Inactivación de Genes , Glicerol Quinasa/genética , Glicosilación , Fenotipo , Regiones Promotoras Genéticas , Procesamiento Proteico-Postraduccional , ARN Polimerasa III/genética , Transcriptoma
9.
ACS Synth Biol ; 9(9): 2515-2524, 2020 09 18.
Artículo en Inglés | MEDLINE | ID: mdl-32786350

RESUMEN

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.


Asunto(s)
Secuenciación de Inmunoprecipitación de Cromatina/métodos , ADN Intergénico/genética , Sistemas CRISPR-Cas/genética , Edición Génica , Factor Estimulante de Colonias de Granulocitos/genética , Factor Estimulante de Colonias de Granulocitos/metabolismo , Hormona de Crecimiento Humana/genética , Hormona de Crecimiento Humana/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo
10.
Nat Biotechnol ; 2018 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-30272677

RESUMEN

Conventional manufacturing of protein biopharmaceuticals in centralized, large-scale, single-product facilities is not well-suited to the agile production of drugs for small patient populations or individuals. Previous solutions for small-scale manufacturing are limited in both process reproducibility and product quality, owing to their complicated means of protein expression and purification. We describe an automated, benchtop, multiproduct manufacturing system, called Integrated Scalable Cyto-Technology (InSCyT), for the end-to-end production of hundreds to thousands of doses of clinical-quality protein biologics in about 3 d. Unlike previous systems, InSCyT includes fully integrated modules for sustained production, efficient purification without the use of affinity tags, and formulation to a final dosage form of recombinant biopharmaceuticals. We demonstrate that InSCyT can accelerate process development from sequence to purified drug in 12 weeks. We used integrated design to produce human growth hormone, interferon α-2b and granulocyte colony-stimulating factor with highly similar processes on this system and show that their purity and potency are comparable to those of marketed reference products.

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