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1.
Int J Biol Macromol ; 180: 760-770, 2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-33716129

RESUMO

Streptokinase is a well-established cost-effective therapeutic molecule for thrombo-embolic complications. In the current study, a tag-free variant of streptokinase with a native N-terminus (N-rSK) was developed using the Pichia expression system. A three-copy clone was screened that secreted 1062 mg/L of N-rSK in the complex medium at shake flask level. The biologically active (67,552.61 IU/mg) N-rSK recovered by anion exchange chromatography was predicted to contain 15.43% α-helices, 26.43% ß-sheets. The fermentation run in a complex medium yielded a poor quality product due to excessive N-rSK degradation. Therefore, modified basal salt medium was also employed during fermentation operations to reduce the proteolytic processing of the recombinant product. The concomitant feeding of 1 g/L/h soya flour hydrolysate with methanol during the protein synthesis phase reduced the proteolysis and yielded 2.29 g/L of N-rSK. The fermentation medium was also supplemented with urea during growth and induction phases. The combined feeding approach of nitrogen-rich soya flour hydrolysate and urea during bioreactor operations showed significant improvement in protein stability and resulted in a 4-fold increase in N-rSK concentration to a level of 4.03 g/L over shake flask. Under optimized conditions, the volumetric productivity and specific product yield were 52.33 mg/L/h and 33.24 mg/g DCW, respectively.


Assuntos
Reatores Biológicos/microbiologia , Fermentação/efeitos dos fármacos , Nitrogênio/farmacologia , Saccharomycetales/metabolismo , Streptococcus/enzimologia , Estreptoquinase/metabolismo , Técnicas de Cultura Celular por Lotes/métodos , Técnicas de Cultura Celular por Lotes/normas , Eletroforese em Gel de Poliacrilamida , Regulação Enzimológica da Expressão Gênica , Nitrogênio/metabolismo , Proteólise , Controle de Qualidade , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Saccharomycetales/citologia , Saccharomycetales/genética , Streptococcus/genética , Estreptoquinase/genética
2.
Biotechnol Prog ; 37(1): e3074, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32865874

RESUMO

Spectroscopy techniques are being implemented within the biopharmaceutical industry due to their non-destructive ability to measure multiple analytes simultaneously, however, minimal work has been applied focussing on their application at small scale. Miniature bioreactor systems are being applied across the industry for cell line development as they offer a high-throughput solution for screening and process optimization. The application of small volume, high-throughput, automated analyses to miniature bioreactors has the potential to significantly augment the type and quality of data from these systems and enhance alignment with large-scale bioreactors. Here, we present an evaluation of 1. a prototype that fully integrates spectroscopy to a miniature bioreactor system (ambr®15, Sartorius Stedim Biotech) enabling automated Raman spectra acquisition, 2. In 50 L single-use bioreactor bag (SUB) prototype with an integrated spectral window. OPLS models were developed demonstrating good accuracy for multiple analytes at both scales. Furthermore, the 50 L SUB prototype enabled on-line monitoring without the need for sterilization of the probe prior to use and minimal light interference was observed. We also demonstrate the ability to build robust models due to induced changes that are hard and costly to perform at large scale and the potential of transferring these models across the scales. The implementation of this technology enables integration of spectroscopy at the small scale for better process understanding and generation of robust models over a large design space while facilitating model transfer throughout the scales enabling continuity throughout process development and utilization and transfer of ever-increasing data generation from development to manufacturing.


Assuntos
Técnicas de Cultura Celular por Lotes/normas , Reatores Biológicos/normas , Ensaios de Triagem em Larga Escala/métodos , Análise Espectral Raman/métodos , Animais , Células CHO , Cricetinae , Cricetulus , Imunoglobulina G/análise
3.
Molecules ; 25(23)2020 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-33266156

RESUMO

Production of monoclonal antibodies (mAbs) is a well-known method used to synthesize a large number of identical antibodies, which are molecules of huge importance in medicine. Due to such reasons, intense efforts have been invested to maximize the mAbs production in bioreactors with hybridoma cell cultures. However, the optimal control of such sensitive bioreactors is an engineering problem difficult to solve due to the large number of state-variables with highly nonlinear dynamics, which often translates into a non-convex optimization problem that involves a significant number of decision (control) variables. Based on an adequate kinetic model adopted from the literature, this paper focuses on developing an in-silico (model-based, offline) numerical analysis of a fed-batch bioreactor (FBR) with an immobilized hybridoma culture to determine its optimal feeding policy by considering a small number of control variables, thus ensuring maximization of mAbs production. The obtained time stepwise optimal feeding policies of FBR were proven to obtain better performances than those of simple batch operation (BR) for all the verified alternatives in terms of raw material consumption and mAbs productivity. Several elements of novelty (i-iv) are pointed out in the "conclusions" section (e.g., considering the continuously added biomass as a control variable during FBR).


Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/isolamento & purificação , Técnicas de Cultura Celular por Lotes/métodos , Técnicas de Cultura Celular por Lotes/normas , Reatores Biológicos/normas , Hibridomas/citologia , Modelos Biológicos , Animais , Cinética , Camundongos
4.
Front Immunol ; 11: 593363, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33613517

RESUMO

Dendritic cell (DC)-based antitumor vaccines have proven to be a safe approach, but often fail to generate robust results between trials. Translation to the clinic has been hindered in part by the lack of standard operation procedures for vaccines production, namely the definition of optimal culture conditions during ex-vivo DC differentiation. Here we sought to compare the ability of three clinical grade serum-free media, DendriMACS, AIM-V, and X-VIVO 15, alongside with fetal bovine serum-supplemented Roswell Park Memorial Institute Medium (RPMI), to support the differentiation of monocyte-derived DCs (Mo-DCs). Under these different culture conditions, phenotype, cell metabolomic profiles, response to maturation stimuli, cytokines production, allogenic T cell stimulatory capacity, as well as priming of antigen-specific CD8+ T cells and activation of autologous natural killer (NK) cells were analyzed. Immature Mo-DCs differentiated in AIM-V or X-VIVO 15 presented lower levels of CD1c, CD1a, and higher expression of CD11c, when compared to cells obtained with DendriMACS. Upon stimulation, only AIM-V or X-VIVO 15 DCs acquired a full mature phenotype, which supports their enhanced capacity to polarize T helper cell type 1 subset, to prime antigen-specific CD8+ T cells and to activate NK cells. CD8+ T cells and NK cells resulting from co-culture with AIM-V or X-VIVO 15 DCs also showed superior cytolytic activity. 1H nuclear magnetic resonance-based metabolomic analysis revealed that superior DC immunostimulatory capacities correlate with an enhanced catabolism of amino acids and glucose. Overall, our data highlight the impact of critically defining the culture medium used in the production of DCs for clinical application in cancer immunotherapy. Moreover, the manipulation of metabolic state during differentiation could be envisaged as a strategy to enhance desired cell characteristics.


Assuntos
Técnicas de Cultura Celular por Lotes , Meios de Cultura Livres de Soro , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Imunoterapia , Cultura Primária de Células/métodos , Técnicas de Cultura Celular por Lotes/métodos , Técnicas de Cultura Celular por Lotes/normas , Biomarcadores , Diferenciação Celular , Citocinas/metabolismo , Testes Imunológicos de Citotoxicidade , Células Dendríticas/citologia , Humanos , Imunofenotipagem , Imunoterapia/métodos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Ativação Linfocitária/imunologia , Neoplasias/imunologia , Neoplasias/patologia , Neoplasias/terapia , Fagocitose , Cultura Primária de Células/normas
5.
Blood Adv ; 3(21): 3337-3350, 2019 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-31698463

RESUMO

Transfusion of donor-derived red blood cells (RBC) is the most common form of cellular therapy. Donor availability and the potential risk of alloimmunization and other transfusion-related complications may, however, limit the availability of transfusion units, especially for chronically transfused patients. In vitro cultured, customizable RBC would negate these concerns and further increase precision medicine. Large-scale, cost-effective production depends on optimization of culture conditions. We developed a defined medium and adapted our protocols to good manufacturing practice (GMP) culture requirements, which reproducibly provided pure erythroid cultures from peripheral blood mononuclear cells without prior CD34+ isolation, and a 3 × 107-fold increase in erythroblasts in 25 days (or from 100 million peripheral blood mononuclear cells, 2 to 4 mL packed red cells can be produced). Expanded erythroblast cultures could be differentiated to CD71dimCD235a+CD44+CD117-DRAQ5- RBC in 12 days. More than 90% of the cells enucleated and expressed adult hemoglobin as well as the correct blood group antigens. Deformability and oxygen-binding capacity of cultured RBC was comparable to in vivo reticulocytes. Daily RNA sampling during differentiation followed by RNA-sequencing provided a high-resolution map/resource of changes occurring during terminal erythropoiesis. The culture process was compatible with upscaling using a G-Rex bioreactor with a capacity of 1 L per reactor, allowing transition toward clinical studies and small-scale applications.


Assuntos
Técnicas de Cultura Celular por Lotes , Diferenciação Celular , Eritroblastos/citologia , Eritrócitos/citologia , Eritropoese , Leucócitos Mononucleares/citologia , Técnicas de Cultura Celular por Lotes/normas , Biomarcadores , Reatores Biológicos , Diferenciação Celular/genética , Proliferação de Células , Eritroblastos/metabolismo , Eritrócitos/metabolismo , Eritropoese/genética , Perfilação da Expressão Gênica , Humanos , Imunofenotipagem , Leucócitos Mononucleares/metabolismo , Cultura Primária de Células , Reticulócitos/metabolismo , Transcriptoma
6.
Biotechnol J ; 14(2): e1700721, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30024096

RESUMO

Due to the growing interest in integrated continuous processing in the biopharmaceutical industry, productivity comparison of batch-based and continuous processes is considered a challenge. Integrated continuous manufacturing of biopharmaceuticals requires scientists and engineers to collaborate effectively. Differing definitions, for example, of volumetric productivity, may cause confusion in this interdisciplinary field. Therefore, the aim of this communication is to reiterate the standard definitions and their underlying assumptions. Applying them to an exemplary model scenario allows to demonstrate the differences and to develop recommendations for the comparison of productivity of different upstream processes.


Assuntos
Técnicas de Cultura Celular por Lotes/métodos , Técnicas de Cultura Celular por Lotes/normas , Biofarmácia/normas , Reatores Biológicos/normas , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/química , Técnicas de Cultura Celular por Lotes/economia , Biofarmácia/economia , Reatores Biológicos/economia , Células CHO , Contagem de Células , Cricetulus , Meios de Cultura , Modelos Teóricos
7.
BioDrugs ; 32(5): 405-414, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30143982

RESUMO

Biologics are produced from living organisms in complex, multi-stage manufacturing processes and contain inherent variability, which must be understood and controlled during manufacturing to avoid unexpected changes in key quality attributes that may contribute to clinically meaningful differences. The process must also meet large commercial demand, while simultaneously being able to accommodate change without sacrificing product consistency. The four key components of successful biologics manufacturing are (1) a stable, well-defined proprietary cell line; (2) a good manufacturing practice (GMP)-compliant supply chain with a process control strategy defining acceptable levels of variability for target product/process attributes and capable of managing complex material flows; (3) a tightly controlled procedure for implementation of proposed process changes that ensures product consistency; and (4) built-in redundancy and flexibility providing the ability to adapt rapidly to unexpected developments. This report describes the requirements for the manufacturing and distribution of biologics, using Remicade® (infliximab, Janssen Biotech, Horsham, PA, USA) as an example of best practices. Since Remicade's first marketing approval in 1998, Janssen has manufactured > 150 million vials used to treat > 2.6 million patients around the world for a variety of inflammatory diseases. Remicade displays a highly consistent quality attribute profile and meets all product/process specifications across multiple manufacturing sites and process scales. Janssen's experience with Remicade demonstrates that deep product knowledge, extensive manufacturing experience, diligent product/process monitoring and a sustained commitment to compliance and research are required to ensure quality, consistency and uninterrupted patient supply for large-volume biologics over the long term.


Assuntos
Produtos Biológicos/provisão & distribuição , Indústria Farmacêutica/normas , Infliximab , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/metabolismo , Técnicas de Cultura Celular por Lotes/métodos , Técnicas de Cultura Celular por Lotes/normas , Produtos Biológicos/normas , Linhagem Celular , Indústria Farmacêutica/métodos , Rotulagem de Medicamentos/normas , Armazenamento de Medicamentos , Liofilização , Controle de Qualidade
8.
Front Immunol ; 9: 1062, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29899740

RESUMO

γ9δ2T cells play a critical role in daily cancer immune surveillance by sensing cancer-mediated metabolic changes. However, a major limitation of the therapeutic application of γ9δ2T cells is their diversity and regulation through innate co-receptors. In order to overcome natural obstacles of γ9δ2T cells, we have developed the concept of T cells engineered to express a defined γδT cell receptor (TEGs). This next generation of chimeric antigen receptor engineered T (CAR-T) cells not only allows for targeting of hematological but also of solid tumors and, therefore, overcomes major limitations of many CAR-T and γδT cell strategies. Here, we report on the development of a robust manufacturing procedure of T cells engineered to express the high affinity Vγ9Vδ2T cell receptor (TCR) clone 5 (TEG001). We determined the best concentration of anti-CD3/CD28 activation and expansion beads, optimal virus titer, and cell density for retroviral transduction, and validated a Good Manufacturing Practice (GMP)-grade purification procedure by utilizing the CliniMACS system to deplete non- and poorly-engineered T cells. To the best of our knowledge, we have developed the very first GMP manufacturing procedure in which αßTCR depletion is used as a purification method, thereby delivering untouched clinical grade engineered immune cells. This enrichment method is applicable to any engineered T cell product with a reduced expression of endogenous αßTCRs. We report on release criteria and the stability of TEG001 drug substance and TEG001 drug product. The GMP-grade production procedure is now approved by Dutch authorities and allows TEG001 to be generated in cell numbers sufficient to treat patients within the approved clinical trial NTR6541. NTR6541 will investigate the safety and tolerability of TEG001 in patients with relapsed/refractory acute myeloid leukemia, high-risk myelodysplastic syndrome, and relapsed/refractory multiple myeloma.


Assuntos
Técnicas de Cultura Celular por Lotes , Expressão Gênica , Engenharia Genética , Receptores de Antígenos de Linfócitos T gama-delta/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Técnicas de Cultura Celular por Lotes/métodos , Técnicas de Cultura Celular por Lotes/normas , Biomarcadores , Técnicas de Cultura de Células , Linhagem Celular , Citotoxicidade Imunológica , Ensaio de Imunoadsorção Enzimática , Vetores Genéticos/genética , Humanos , Imunofenotipagem , Imunoterapia Adotiva/métodos , Imunoterapia Adotiva/normas , Ativação Linfocitária/imunologia , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/terapia , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Transdução Genética , Transgenes
9.
Adv Biochem Eng Biotechnol ; 165: 443-462, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29721583

RESUMO

As biopharmaceutical companies have optimized cell line and production culture process development, titers of recombinant antibodies have risen steadily to 3-8 g/L for fed-batch mammalian cultures at production scales of 10 kL or larger. Most new antibody products are produced from Chinese Hamster Ovary (CHO) cell lines, and there are relatively few alternative production hosts under active evaluation. Many companies have adopted a strategy of using the same production cell line for early clinical phases as well as commercial production, which reduces the risk of product comparability issues during the development lifecycle. Product quality and consistency expectations rest on the platform knowledge of the CHO host cell line and processes used for the production of many licensed antibodies. The lack of impact of low-level product variants common to this platform on product safety and efficacy also builds on the established commercial history of recombinant antibodies, which dates back to 1997.Efforts to increase titers further will likely yield diminishing returns. Very few products would benefit significantly from a titer greater than 8 g/L; in many cases, a downstream processing bottleneck would preclude full recovery from production-scale bioreactors for high titer processes. The benefits of a process platform based on standard fed-batch production culture include predictable scale-up, process transfer, and production within a company's manufacturing network or at a contract manufacturing organization. Furthermore, the confidence in an established platform provides key support towards regulatory flexibility (e.g., design space) for license applications following a quality-by-design strategy.These factors suggest that novel technologies for antibody production may not provide a substantial return on investment. What, then, should be the focus of future process development efforts for companies that choose to launch antibody products using their current platform? This review proposes key focus areas in an effort to continually improve process consistency, assure acceptable product quality, and establish appropriate process parameter limits to enable flexible manufacturing options.


Assuntos
Anticorpos Monoclonais , Técnicas de Cultura Celular por Lotes , Animais , Técnicas de Cultura Celular por Lotes/normas , Técnicas de Cultura Celular por Lotes/tendências , Reatores Biológicos , Células CHO , Cricetinae , Cricetulus
10.
Biotechnol Lett ; 40(7): 1029-1036, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29693210

RESUMO

OBJECTIVES: Establish a method to indirectly measure evaporation in microwell-based cell culture systems and show that the proposed method allows compensating for liquid losses in fed-batch processes. RESULTS: A correlation between evaporation and the concentration of Na+ was found (R2 = 0.95) when using the 24-well-based miniature bioreactor system (micro-Matrix) for a batch culture with GS-CHO. Based on these results, a method was developed to counteract evaporation with periodic water additions based on measurements of the Na+ concentration. Implementation of this method resulted in a reduction of the relative liquid loss after 15 days of a fed-batch cultivation from 36.7 ± 6.7% without volume corrections to 6.9 ± 6.5% with volume corrections. CONCLUSION: A procedure was established to indirectly measure evaporation through a correlation with the level of Na+ ions in solution and deriving a simple formula to account for liquid losses.


Assuntos
Técnicas de Cultura Celular por Lotes/métodos , Técnicas de Cultura Celular por Lotes/normas , Reatores Biológicos/normas , Água , Animais , Células CHO , Fenômenos Químicos , Cricetinae , Cricetulus , Reprodutibilidade dos Testes , Sódio/análise , Sódio/química , Água/análise , Água/química
11.
Artigo em Inglês | MEDLINE | ID: mdl-29596027

RESUMO

Despite the fact that biological nitrogen removal (BNR) process has been studied in detail in laboratory- and pilot-scale sequencing batch reactor (SBR) systems treating landfill leachate, a limited number of research works have been performed in full-scale SBR plants regarding nitrification and denitrification. In the current study, a full-scale twin SBR system in series of 700 m3 (350 m3 each) treating medium-age landfill leachate was evaluated in terms of its carbon and nitrogen removal efficiency in the absence and presence of external carbon source, i.e., glycerol from biodiesel production. Both biodegradable organic carbon and ammonia were highly oxidized [biochemical oxygen demand (BOD5) and total Kjehldahl nitrogen (TKN) removal efficiencies above 90%], whereas chemical oxygen demand (COD) removal efficiency was slightly above 40%, which is within the range reported in the literature for pilot-scale SBRs. As the consequence of the high recalcitrant organic fraction of the landfill leachate, dissimilatory nitrate reduction was restricted in the absence of crude glycerol, although denitrification was improved by electron donor addition, resulting in TN removal efficiencies above 70%. Experimental data revealed that the second SBR negligibly contributed to BNR process, since carbon and ammonia oxidation completion was achieved in the first SBR. On the other hand, the low VSS/SS ratio, due to the lack of primary sedimentation, highly improved sludge settleability, resulting in sludge volume indices (SVI) below 30 mL g-1.


Assuntos
Técnicas de Cultura Celular por Lotes , Reatores Biológicos , Desnitrificação , Nitrogênio/isolamento & purificação , Poluentes Químicos da Água/isolamento & purificação , Amônia/química , Técnicas de Cultura Celular por Lotes/instrumentação , Técnicas de Cultura Celular por Lotes/métodos , Técnicas de Cultura Celular por Lotes/normas , Análise da Demanda Biológica de Oxigênio , Reatores Biológicos/normas , Calibragem , Carbono/química , Humanos , Nitrificação , Nitrogênio/química , Nitrogênio/farmacocinética , Esgotos/química , Instalações de Eliminação de Resíduos , Poluentes Químicos da Água/química , Poluentes Químicos da Água/farmacocinética
12.
Biotechnol Prog ; 33(6): 1647-1661, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28786215

RESUMO

As the biopharmaceutical industry evolves to include more diverse protein formats and processes, more robust control of Critical Quality Attributes (CQAs) is needed to maintain processing flexibility without compromising quality. Active control of CQAs has been demonstrated using model predictive control techniques, which allow development of processes which are robust against disturbances associated with raw material variability and other potentially flexible operating conditions. Wide adoption of model predictive control in biopharmaceutical cell culture processes has been hampered, however, in part due to the large amount of data and expertise required to make a predictive model of controlled CQAs, a requirement for model predictive control. Here we developed a highly automated, perfusion apparatus to systematically and efficiently generate predictive models using application of system identification approaches. We successfully created a predictive model of %galactosylation using data obtained by manipulating galactose concentration in the perfusion apparatus in serialized step change experiments. We then demonstrated the use of the model in a model predictive controller in a simulated control scenario to successfully achieve a %galactosylation set point in a simulated fed-batch culture. The automated model identification approach demonstrated here can potentially be generalized to many CQAs, and could be a more efficient, faster, and highly automated alternative to batch experiments for developing predictive models in cell culture processes, and allow the wider adoption of model predictive control in biopharmaceutical processes. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 33:1647-1661, 2017.


Assuntos
Anticorpos Monoclonais/biossíntese , Técnicas de Cultura Celular por Lotes/normas , Biofarmácia/normas , Reatores Biológicos/normas , Animais , Anticorpos Monoclonais/química , Técnicas de Cultura Celular por Lotes/métodos , Biofarmácia/métodos , Células CHO , Cricetinae , Cricetulus , Humanos , Controle de Qualidade
13.
Biotechnol Bioeng ; 114(7): 1448-1458, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28197999

RESUMO

Rational and high-throughput optimization of mammalian cell culture media has a great potential to modulate recombinant protein product quality. We present a process design method based on parallel design-of-experiment (DoE) of CHO fed-batch cultures in 96-deepwell plates to modulate monoclonal antibody (mAb) glycosylation using medium supplements. To reduce the risk of losing valuable information in an intricate joint screening, 17 compounds were separated into five different groups, considering their mode of biological action. The concentration ranges of the medium supplements were defined according to information encountered in the literature and in-house experience. The screening experiments produced wide glycosylation pattern ranges. Multivariate analysis including principal component analysis and decision trees was used to select the best performing glycosylation modulators. Subsequent D-optimal quadratic design with four factors (three promising compounds and temperature shift) in shake tubes confirmed the outcome of the selection process and provided a solid basis for sequential process development at a larger scale. The glycosylation profile with respect to the specifications for biosimilarity was greatly improved in shake tube experiments: 75% of the conditions were equally close or closer to the specifications for biosimilarity than the best 25% in 96-deepwell plates. Biotechnol. Bioeng. 2017;114: 1448-1458. © 2017 Wiley Periodicals, Inc.


Assuntos
Técnicas de Cultura Celular por Lotes/métodos , Medicamentos Biossimilares/metabolismo , Meios de Cultura/química , Meios de Cultura/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Proteínas Recombinantes/biossíntese , Análise Serial de Tecidos/métodos , Animais , Anticorpos Monoclonais , Técnicas de Cultura Celular por Lotes/normas , Medicamentos Biossimilares/normas , Células CHO , Cricetulus , Meios de Cultura/normas , Ensaios de Triagem em Larga Escala/normas , Análise Multivariada , Análise de Componente Principal , Engenharia de Proteínas/métodos , Engenharia de Proteínas/normas , Controle de Qualidade , Proteínas Recombinantes/normas
14.
PDA J Pharm Sci Technol ; 71(3): 189-205, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27974629

RESUMO

In order to operate profitably under different product demand scenarios, biopharmaceutical companies must design their facilities with mass output flexibility in mind. Traditional biologics manufacturing technologies pose operational challenges in this regard due to their high costs and slow equipment turnaround times, restricting the types of products and mass quantities that can be processed. Modern plant design, however, has facilitated the development of lean and efficient bioprocessing facilities through footprint reduction and adoption of disposable and continuous manufacturing technologies. These development efforts have proven to be crucial in seeking to drastically reduce the high costs typically associated with the manufacturing of recombinant proteins. In this work, mathematical modeling is used to optimize annual production schedules for a single-product commercial facility operating with a continuous upstream and discrete batch downstream platform. Utilizing cell culture duration and volumetric productivity as process variables in the model, and annual plant throughput as the optimization objective, 3-D surface plots are created to understand the effect of process and facility design on expected mass output. The model shows that once a plant has been fully debottlenecked it is capable of processing well over a metric ton of product per year. Moreover, the analysis helped to uncover a major limiting constraint on plant performance, the stability of the neutralized viral inactivated pool, which may indicate that this should be a focus of attention during future process development efforts.LAY ABSTRACT: Biopharmaceutical process modeling can be used to design and optimize manufacturing facilities and help companies achieve a predetermined set of goals. One way to perform optimization is by making the most efficient use of process equipment in order to minimize the expenditure of capital, labor and plant resources. To that end, this paper introduces a novel mathematical algorithm used to determine the most optimal equipment scheduling configuration that maximizes the mass output for a facility producing a single product. The paper also illustrates how different scheduling arrangements can have a profound impact on the availability of plant resources, and identifies limiting constraints on the plant design. In addition, simulation data is presented using visualization techniques that aid in the interpretation of the scientific concepts discussed.


Assuntos
Produtos Biológicos/química , Biofarmácia/normas , Indústria Farmacêutica/normas , Arquitetura de Instituições de Saúde , Preparações Farmacêuticas/química , Controle de Qualidade , Tecnologia Farmacêutica/normas , Fluxo de Trabalho , Técnicas de Cultura Celular por Lotes/normas , Biofarmácia/instrumentação , Biofarmácia/métodos , Reatores Biológicos/normas , Cromatografia/normas , Composição de Medicamentos , Indústria Farmacêutica/instrumentação , Indústria Farmacêutica/métodos , Filtração/normas , Modelos Teóricos , Tecnologia Farmacêutica/instrumentação , Tecnologia Farmacêutica/métodos , Fatores de Tempo
15.
Exp Hematol ; 45: 27-35.e1, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27693388

RESUMO

Recent clinical trials have indicated the high potential of regulatory T cells (Tregs) in the prevention of acute and chronic graft-versus-host disease (GvHD) after hematopoietic stem cell transplantation, but immune interventions require large numbers of Tregs. With respect to their limited natural occurrence, development and optimization of protocols for large-scale expansion of clinical-grade Tregs are essential if considered for therapeutic use. We compared different clinical-grade large-scale expansion protocols for repetitive transfer of large numbers of Tregs in clinical trials for the prevention of acute and/or chronic GvHD. Donor Tregs were isolated using magnetic-activated cell sorting (MACS) technology with good manufacturing practice-compliant devices. CD8 and CD19 depletion followed by CD25 enrichment resulted in the isolation of CD4+CD25+CD127- Tregs with a mean purity of 77%. Cell populations were expanded ex vivo using X-Vivo 15 (±rapamycin), TexMACS (±rapamycin), and CellGro DC (±rapamycin) in the presence of interleukin-2. The highest rates of expansion of clinical-grade Tregs were observed for X-Vivo 15 and CellGro DC without rapamycin in compared with all other expansion media tested. The suppressive capacity of the expanded Treg population was maintained under all conditions investigated. Our data suggest that expansion with CellGro provides data comparable to those obtained with TexMACS or X-Vivo 15 with rapamycin, although all three conditions did not provide the same propagation rate as X-Vivo 15 alone. With respect to functionality, phenotype, and stability, CellGro DC medium represents a reasonable alternative for good manufacturing practice-compatible large-scale ex vivo expansion.


Assuntos
Transferência Adotiva/métodos , Transferência Adotiva/normas , Técnicas de Cultura Celular por Lotes , Fidelidade a Diretrizes , Linfócitos T Reguladores , Adulto , Técnicas de Cultura Celular por Lotes/métodos , Técnicas de Cultura Celular por Lotes/normas , Biomarcadores , Movimento Celular , Separação Celular , Metilação de DNA , Feminino , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/terapia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Imunofenotipagem , Terapia de Imunossupressão , Masculino , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Adulto Jovem
16.
Biotechnol Bioeng ; 114(4): 785-797, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-27869296

RESUMO

We describe a systematic approach to model CHO metabolism during biopharmaceutical production across a wide range of cell culture conditions. To this end, we applied the metabolic steady state concept. We analyzed and modeled the production rates of metabolites as a function of the specific growth rate. First, the total number of metabolic steady state phases and the location of the breakpoints were determined by recursive partitioning. For this, the smoothed derivative of the metabolic rates with respect to the growth rate were used followed by hierarchical clustering of the obtained partition. We then applied a piecewise regression to the metabolic rates with the previously determined number of phases. This allowed identifying the growth rates at which the cells underwent a metabolic shift. The resulting model with piecewise linear relationships between metabolic rates and the growth rate did well describe cellular metabolism in the fed-batch cultures. Using the model structure and parameter values from a small-scale cell culture (2 L) training dataset, it was possible to predict metabolic rates of new fed-batch cultures just using the experimental specific growth rates. Such prediction was successful both at the laboratory scale with 2 L bioreactors but also at the production scale of 2000 L. This type of modeling provides a flexible framework to set a solid foundation for metabolic flux analysis and mechanistic type of modeling. Biotechnol. Bioeng. 2017;114: 785-797. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.


Assuntos
Anticorpos Monoclonais/análise , Anticorpos Monoclonais/metabolismo , Técnicas de Cultura Celular por Lotes/métodos , Técnicas de Cultura Celular por Lotes/normas , Reatores Biológicos , Modelos Lineares , Animais , Células CHO , Calibragem , Cricetinae , Cricetulus , Reprodutibilidade dos Testes
17.
J Biosci Bioeng ; 122(5): 563-569, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27209176

RESUMO

Ethanol is a very important industrial chemical. In order to improve ethanol productivity using Saccharomyces cerevisiae in fermentation from furfural process residue, we developed a process of simultaneous saccharification and fermentation (SSF) of furfural process residue, optimizing prehydrolysis cellulase loading concentration, prehydrolysis time, and substrate feeding strategy. The ethanol concentration obtained from the optimized process was 19.3 g/L, corresponding 76.5% ethanol yield, achieved by running SSF for 48 h from 10% furfural process residue with prehydrolysis at 50°C for 4 h and cellulase loading of 15 FPU/g furfural process residue. For higher ethanol concentrations, fed-batch fermentation was performed. The optimized fed-batch process increased the ethanol concentration to 37.6 g/L, 74.5% yield, obtained from 10% furfural process residue with two additions of 5% substrate at 12 and 24 h.


Assuntos
Técnicas de Cultura Celular por Lotes/normas , Etanol/metabolismo , Fermentação , Furaldeído/metabolismo , Saccharomyces cerevisiae , Sacarose/metabolismo , Técnicas de Cultura Celular por Lotes/métodos , Calibragem , Celulase/metabolismo , Hidrólise , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Fatores de Tempo
18.
Hum Gene Ther Methods ; 26(6): 197-210, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26467420

RESUMO

We evaluated the possibility of introducing a combination of six oncogenes into primary porcine hepatocytes (PPH) using a lentiviral vector (LV)-mediated gene transfer in order to develop a porcine hepatocellular carcinoma model based on autologous transplantation of ex vivo-transformed hepatocytes. The six oncogenes were introduced into three plasmids, hence enabling the production of LVs encoding a luciferase reporter gene and hTERT+p53(DD), cyclinD1+CDK4(R24C), and c-myc(T58A)+HRas(G21V) genes, respectively. In order to improve the protection of the laboratory personnel manipulating such LVs, we used a compact cell culture cassette (CliniCell(®) device) as a closed cell culture system. We demonstrated that the CliniCell device allows to produce LVs, through plasmid transfection of 293T cells, and, after transfer to a second cassette, to transduce PPH with a similar efficacy as conventional open cell culture systems such as flasks or Petri dishes. Additionally, it is possible to cryopreserve at -80°C the transduced cells, directly in the CliniCell device used for the transduction. In conclusion, the use of a closed culture system for the safe handling of oncogene-encoding LVs lays the foundation for the development of porcine tumor models based on the autologous transplantation of ex vivo-transformed primary cells.


Assuntos
Técnicas de Cultura Celular por Lotes , Vetores Genéticos/genética , Vetores Genéticos/isolamento & purificação , Lentivirus/genética , Replicação Viral , Animais , Técnicas de Cultura Celular por Lotes/instrumentação , Técnicas de Cultura Celular por Lotes/métodos , Técnicas de Cultura Celular por Lotes/normas , Expressão Gênica , Genes Reporter , Células HEK293 , Hepatócitos , Humanos , Suínos , Transdução Genética , Transgenes
19.
J Immunother ; 38(3): 127-35, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25751502

RESUMO

The successful genetic engineering of patient T cells with γ-retroviral vectors expressing chimeric antigen receptors or T-cell receptors for phase II clinical trials and beyond requires the large-scale manufacture of high-titer vector stocks. The production of retroviral vectors from stable packaging cell lines using roller bottles or 10- to 40-layer cell factories is limited by a narrow harvest window, labor intensity, open-system operations, and the requirement for significant incubator space. To circumvent these shortcomings, we optimized the production of vector stocks in a disposable fixed-bed bioreactor using good manufacturing practice-grade packaging cell lines. High-titer vector stocks were harvested over 10 days, representing a much broader harvest window than the 3-day harvest afforded by cell factories. For PG13 and 293Vec packaging cells, the average vector titer and the vector stocks' yield in the bioreactor were higher by 3.2- to 7.3-fold, and 5.6- to 13.1-fold, respectively, than those obtained in cell factories. The vector production was 10.4 and 18.6 times more efficient than in cell factories for PG13 and 293Vec cells, respectively. Furthermore, the vectors produced from the fixed-bed bioreactors passed the release test assays for clinical applications. Therefore, a single vector lot derived from 293Vec is suitable to transduce up to 500 patients cell doses in the context of large clinical trials using chimeric antigen receptors or T-cell receptors. These findings demonstrate for the first time that a robust fixed-bed bioreactor process can be used to produce γ-retroviral vector stocks scalable up to the commercialization phase.


Assuntos
Técnicas de Cultura Celular por Lotes/métodos , Técnicas de Cultura Celular por Lotes/normas , Reatores Biológicos , Vetores Genéticos/genética , Vetores Genéticos/normas , Retroviridae/genética , Animais , Linhagem Celular Transformada , Células HEK293 , Humanos , Vírus da Leucemia do Macaco Gibão/genética , Linfócitos T/metabolismo , Transdução Genética
20.
Biotechnol Bioeng ; 112(3): 536-48, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25219609

RESUMO

The global bio-manufacturing industry requires improved process efficiency to satisfy the increasing demands for biochemicals, biofuels, and biologics. The use of model-based techniques can facilitate the reduction of unnecessary experimentation and reduce labor and operating costs by identifying the most informative experiments and providing strategies to optimize the bioprocess at hand. Herein, we investigate the potential of a research methodology that combines model development, parameter estimation, global sensitivity analysis, and selection of optimal feeding policies via dynamic optimization methods to improve the efficiency of an industrially relevant bioprocess. Data from a set of batch experiments was used to estimate values for the parameters of an unstructured model describing monoclonal antibody (mAb) production in GS-NS0 cell cultures. Global Sensitivity Analysis (GSA) highlighted parameters with a strong effect on the model output and data from a fed-batch experiment were used to refine their estimated values. Model-based optimization was used to identify a feeding regime that maximized final mAb titer. An independent fed-batch experiment was conducted to validate both the results of the optimization and the predictive capabilities of the developed model. The successful integration of wet-lab experimentation and mathematical model development, analysis, and optimization represents a unique, novel, and interdisciplinary approach that addresses the complicated research and industrial problem of model-based optimization of cell based processes.


Assuntos
Anticorpos Monoclonais/metabolismo , Técnicas de Cultura Celular por Lotes/métodos , Técnicas de Cultura Celular por Lotes/normas , Reatores Biológicos , Glucose/metabolismo , Modelos Biológicos , Amônia/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular , Ácido Glutâmico/metabolismo , Ácido Láctico/metabolismo , Camundongos , Reprodutibilidade dos Testes
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