RESUMEN
Bioreactor parameters can have significant effects on the quantity and quality of biotherapeutics. Monoclonal antibody products have one particularly important critical quality attribute being the distribution of product glycoforms. N-linked glycosylation affects the therapeutic properties of the antibody including effector function, immunogenicity, stability, and clearance rate. Our past work revealed that feeding different amino acids to bioreactors altered the productivity and glycan profiles. To facilitate real-time analysis of bioreactor parameters and the glycosylation of antibody products, we developed an on-line system to pull cell-free samples directly from the bioreactors, chemically process them, and deliver them to a chromatography-mass spectroscopy system for rapid identification and quantification. We were able to successfully monitor amino acid concentration on-line within multiple reactors, evaluate glycans off-line, and extract four principal components to assess the amino acid concentration and glycosylation profile relationship. We found that about a third of the variability in the glycosylation data can be predicted from the amino acid concentration. Additionally, we determined that the third and fourth principal component accounts for 72% of our model's predictive power, with the third component indicated to be positively correlated with latent metabolic processes related to galactosylation. Here we present our work on rapid online spent media amino acid analysis and use the determined trends to collate with glycan time progression, further elucidating the correlation between bioreactor parameters such as amino acid nutrient profiles, and product quality. We believe such approaches may be useful for maximizing efficiency and reducing production costs for biotherapeutics.
Asunto(s)
Aminoácidos , Anticuerpos Monoclonales , Anticuerpos Monoclonales/química , Glicosilación , Aminoácidos/metabolismo , Reactores Biológicos , Polisacáridos/químicaRESUMEN
Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.
Asunto(s)
Anticuerpos Monoclonales/aislamiento & purificación , Reactores Biológicos , Aminoácidos/análisis , Aminoácidos/metabolismo , Animales , Automatización , Células CHO , Cromatografía Líquida de Alta Presión , Cricetinae , Cricetulus , Electroforesis Capilar , Fluorescencia , Glicosilación , Inmunoglobulina G/aislamiento & purificación , Espectrometría de Masas , Peso Molecular , Polisacáridos/metabolismoRESUMEN
Amino acids and glucose consumption, cell growth and monoclonal antibody (mAb) production in mammalian cell culture are key considerations during upstream process and particularly media optimization. Understanding the interrelations and the relevant cellular physiology will provide insight for setting strategy of robust and effective mAb production. The aim of this study was to further our understanding of nutrient consumption metabolism, since this could have significant impact on enhancing mAb titer, cell proliferation, designing feeding strategies, and development of feed media. The nutrient consumption pattern, mAb concentration, and cell growth were analyzed in three sets of cell cultures with media supplementation of glucose, methionine, threonine, tryptophan, and tyrosine. The amino acids metabolism and its impact on cell growth and mAb production during the batch and fed-batch culture were closely analyzed. It was shown that the phenylalanine, tyrosine and tryptophan biosynthesis pathways were significantly altered under different culture conditions with different media. These changes were more apparent in the fed-batch process in which higher mAb titer was observed due to the metabolic changes than mAb titer in the batch process. The pathway analysis approach was well utilized for evaluating the impact on the relevant pathways involved under different cell culture conditions to improve cell growth and mAb titer. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:793-805, 2018.
Asunto(s)
Técnicas de Cultivo de Célula , Medios de Cultivo/química , Ingeniería Metabólica , Animales , Reactores Biológicos , Células Cultivadas , RatonesRESUMEN
Monoclonal antibodies are mainly produced by mammalian cell culture, which due to its complexity, results in a wide range of product variants/isoforms. With the growing implementation of Quality by Design (QbD) and Process Analytical Technology (PAT) in drug manufacturing, monitoring and controlling quality attributes within a predefined range during manufacturing may provide added consistency to product quality. To implement these concepts, more robust analytical tools could reduce the time needed for monitoring quality attributes during upstream processing. The formation of protein aggregates is one such quality attribute that can lead to safety and efficacy issues in the final drug product. Described in this study is a fully automated two-dimensional high performance liquid chromatography (2D-HPLC) method for characterizing protein aggregation of crude in-process bioreactor samples. It combines protein A purification and separation by size exclusion into a single analytical module that has the potential to be employed at-line within a bioprocessing system. This method utilizes a novel in-line fraction collection device allowing for the collection of up to twelve fractions from a single sample or peak which facilitates the subsequent linked analysis of multiple protein peaks of interest in one chromatography module.
Asunto(s)
Anticuerpos Monoclonales , Cromatografía Líquida de Alta Presión/instrumentación , Cromatografía Líquida de Alta Presión/métodos , Anticuerpos Monoclonales/análisis , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/aislamiento & purificación , Cromatografía en Gel/instrumentación , Cromatografía en Gel/métodos , Diseño de Equipo , Agregado de Proteínas , Proteína Estafilocócica A/análisis , Proteína Estafilocócica A/química , Proteína Estafilocócica A/aislamiento & purificaciónRESUMEN
Linkage of upstream cell culture with downstream processing and purification is an aspect of Quality by Design crucial for efficient and consistent production of high quality biopharmaceutical proteins. In a previous Plackett-Burman screening study of parallel bioreactor cultures we evaluated main effects of 11 process variables, such as agitation, sparge rate, feeding regimens, dissolved oxygen set point, inoculation density, supplement addition, temperature, and pH shifts. In this follow-up study, we observed linkages between cell culture process parameters and downstream capture chromatography performance and subsequent antibody attributes. In depth analysis of the capture chromatography purification of harvested cell culture fluid yielded significant effects of upstream process parameters on host cell protein abundance and behavior. A variety of methods were used to characterize the antibody both after purification and buffer formulation. This analysis provided insight in to the significant impacts of upstream process parameters on aggregate formation, impurities, and protein structure. This report highlights the utility of linkage studies in identifying how changes in upstream parameters can impact downstream critical quality attributes. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:163-170, 2017.
Asunto(s)
Anticuerpos Monoclonales/aislamiento & purificación , Reactores Biológicos , Técnicas de Cultivo de Célula/métodos , Cromatografía/métodos , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/química , Biotecnología/métodos , Células CHO , Cricetulus , Concentración de Iones de Hidrógeno , TemperaturaRESUMEN
Genome-scale flux balance analysis (FBA) is a powerful systems biology tool to characterize intracellular reaction fluxes during cell cultures. FBA estimates intracellular reaction rates by optimizing an objective function, subject to the constraints of a metabolic model and media uptake/excretion rates. A dynamic extension to FBA, dynamic flux balance analysis (DFBA), can calculate intracellular reaction fluxes as they change during cell cultures. In a previous study by Read et al. (2013), a series of informed amino acid supplementation experiments were performed on twelve parallel murine hybridoma cell cultures, and this data was leveraged for further analysis (Read et al., Biotechnol Prog. 2013;29:745-753). In order to understand the effects of media changes on the model murine hybridoma cell line, a systems biology approach is applied in the current study. Dynamic flux balance analysis was performed using a genome-scale mouse metabolic model, and multivariate data analysis was used for interpretation. The calculated reaction fluxes were examined using partial least squares and partial least squares discriminant analysis. The results indicate media supplementation increases product yield because it raises nutrient levels extending the growth phase, and the increased cell density allows for greater culture performance. At the same time, the directed supplementation does not change the overall metabolism of the cells. This supports the conclusion that product quality, as measured by glycoform assays, remains unchanged because the metabolism remains in a similar state. Additionally, the DFBA shows that metabolic state varies more at the beginning of the culture but less by the middle of the growth phase, possibly due to stress on the cells during inoculation. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1163-1173, 2016.
Asunto(s)
Aminoácidos/metabolismo , Técnicas de Cultivo de Célula , Modelos Biológicos , Biología de Sistemas , Aminoácidos/química , Animales , Células Cultivadas , Glicosilación , Ratones , Análisis MultivarianteRESUMEN
Formulating appropriate storage conditions for biopharmaceutical proteins is essential for ensuring their stability and thereby their purity, potency, and safety over their shelf-life. Using a model murine IgG3 produced in a bioreactor system, multiple formulation compositions were systematically explored in a DoE design to optimize the stability of a challenging antibody formulation worst case. The stability of the antibody in each buffer formulation was assessed by UV/VIS absorbance at 280 nm and 410 nm and size exclusion high performance liquid chromatography (SEC) to determine overall solubility, opalescence, and aggregate formation, respectively. Upon preliminary testing, acetate was eliminated as a potential storage buffer due to significant visible precipitate formation. An additional 2(4) full factorial DoE was performed that combined the stabilizing effect of arginine with the buffering capacity of histidine. From this final DoE, an optimized formulation of 200 mM arginine, 50 mM histidine, and 100 mM NaCl at a pH of 6.5 was identified to substantially improve stability under long-term storage conditions and after multiple freeze/thaw cycles. Thus, our data highlights the power of DoE based formulation screening approaches even for challenging monoclonal antibody molecules.
Asunto(s)
Anticuerpos Monoclonales/química , Formación de Anticuerpos , Inmunoglobulina G/química , Animales , Anticuerpos Monoclonales/inmunología , Tampones (Química) , Cromatografía Líquida de Alta Presión , Congelación , Concentración de Iones de Hidrógeno , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/inmunología , Ratones , Estabilidad ProteicaRESUMEN
Fermentanomics is an emerging field of research and involves understanding the underlying controlled process variables and their effect on process yield and product quality. Although major advancements have occurred in process analytics over the past two decades, accurate real-time measurement of significant quality attributes for a biotech product during production culture is still not feasible. Researchers have used an amalgam of process models and analytical measurements for monitoring and process control during production. This article focuses on using multivariate data analysis as a tool for monitoring the internal bioreactor dynamics, the metabolic state of the cell, and interactions among them during culture. Quality attributes of the monoclonal antibody product that were monitored include glycosylation profile of the final product along with process attributes, such as viable cell density and level of antibody expression. These were related to process variables, raw materials components of the chemically defined hybridoma media, concentration of metabolites formed during the course of the culture, aeration-related parameters, and supplemented raw materials such as glucose, methionine, threonine, tryptophan, and tyrosine. This article demonstrates the utility of multivariate data analysis for correlating the product quality attributes (especially glycosylation) to process variables and raw materials (especially amino acid supplements in cell culture media). The proposed approach can be applied for process optimization to increase product expression, improve consistency of product quality, and target the desired quality attribute profile.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Técnicas de Cultivo de Célula/métodos , Biología Computacional/métodos , Medios de Cultivo/metabolismo , Fermentación/fisiología , Aminoácidos/análisis , Aminoácidos/química , Aminoácidos/metabolismo , Animales , Anticuerpos Monoclonales/análisis , Medios de Cultivo/análisis , Medios de Cultivo/química , Fermentación/genética , Glicosilación , Hibridomas/metabolismo , Ratones , Análisis Multivariante , Proyectos de InvestigaciónRESUMEN
Consistent high-quality antibody yield is a key goal for cell culture bioprocessing. This endpoint is typically achieved in commercial settings through product and process engineering of bioreactor parameters during development. When the process is complex and not optimized, small changes in composition and control may yield a finished product of less desirable quality. Therefore, changes proposed to currently validated processes usually require justification and are reported to the US FDA for approval. Recently, design-of-experiments-based approaches have been explored to rapidly and efficiently achieve this goal of optimized yield with a better understanding of product and process variables that affect a product's critical quality attributes. Here, we present a laboratory-scale model culture where we apply a Plackett-Burman screening design to parallel cultures to study the main effects of 11 process variables. This exercise allowed us to determine the relative importance of these variables and identify the most important factors to be further optimized in order to control both desirable and undesirable glycan profiles. We found engineering changes relating to culture temperature and nonessential amino acid supplementation significantly impacted glycan profiles associated with fucosylation, ß-galactosylation, and sialylation. All of these are important for monoclonal antibody product quality.
Asunto(s)
Anticuerpos Monoclonales/química , Reactores Biológicos , Técnicas de Cultivo de Célula/métodos , Hibridomas/metabolismo , Inmunoglobulina G/química , Polisacáridos/química , Animales , Anticuerpos Monoclonales/metabolismo , Secuencia de Carbohidratos , Proliferación Celular , Glicosilación , Hibridomas/química , Hibridomas/citología , Inmunoglobulina G/metabolismo , Espectrometría de Masas , Ratones , Datos de Secuencia Molecular , Polisacáridos/metabolismo , TemperaturaRESUMEN
Bioreactor process changes can have a profound effect on the yield and quality of biotechnology products. Mannose-6-phosphate (M6P) glycan content and the enzymatic catalytic kinetic parameters are critical quality attributes (CQAs) of many therapeutic enzymes used to treat lysosomal storage diseases (LSDs). Here, we have evaluated the effect of adding butyrate to bioreactor production cultures of human recombinant ß-glucuronidase produced from CHO-K1 cells, with an emphasis on CQAs. The ß-glucuronidase produced in parallel bioreactors was quantified by capillary electrophoresis, the catalytic kinetic parameters were measured using steady-state analysis, and mannose-6-phosphorylation status was assessed using an M6P-specific single-chain antibody fragment. Using this approach, we found that butyrate treatment increased ß-glucuronidase production up to approximately threefold without significantly affecting the catalytic properties of the enzyme. However, M6P content in ß-glucuronidase was inversely correlated with the increased enzyme production induced by butyrate treatment. This assessment demonstrated that although butyrate dramatically increased ß-glucuronidase production in bioreactors, it adversely impacted the mannose-6-phosphorylation of this LSD therapeutic enzyme. This strategy may have utility in evaluating manufacturing process changes to improve therapeutic enzyme yields and CQAs.
Asunto(s)
Reactores Biológicos , Butiratos/farmacología , Glucuronidasa/biosíntesis , Enfermedades por Almacenamiento Lisosomal/enzimología , Animales , Butiratos/química , Células CHO , Cricetinae , Cricetulus , Glucuronidasa/uso terapéutico , Humanos , Enfermedades por Almacenamiento Lisosomal/tratamiento farmacológico , Enfermedades por Almacenamiento Lisosomal/patología , Manosafosfatos/química , Manosafosfatos/farmacología , Fosforilación , Polisacáridos/químicaRESUMEN
Fermentanomics, or a global understanding of a culture state on the molecular level empowered by advanced techniques like NMR, was employed to show that a model hybridoma culture supplied with glutamine and glucose depletes aspartate, cysteine, methionine, tryptophan, and tyrosine during antibody production. Supplementation with these amino acids prevents depletion and improves culture performance. Furthermore, no significant changes were observed in the distribution of glycans attached to the IgG3 in cultures supplemented with specific amino acids, arguing that this strategy can be implemented without fear of impact on important product quality attributes. In summary, a targeted strategy of quantifying media components and designing a supplementation strategy can improve bioprocess cell cultures when enpowered by fermentanomics tools.